Methods and composition for gene delivery using an engineered viral particle

ABSTRACT

The present invention provides compositions and methods for transducing cells (e.g. T cells or immune cells). Also provided herein are methods of treating a disease in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 17/198,970, filed Mar. 11, 2021, whichissued as U.S. Pat. No. 11,191,784 on Dec. 7, 2021, which claimspriority to U.S. Provisional Application No. 62/988,195 filed Mar. 11,2020, to U.S. Provisional Application No. 62/988,074 filed Mar. 11,2020, and to U.S. Provisional Application No. 63/080,501 filed Sep. 18,2020, each of which is herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing concurrently submitted herewith as a text filenamed “046483_7300US2_Sequence_Listing.txt,” created on Nov. 2, 2021 andhaving a size of 58,000 bytes is herein incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

CAR T cell therapy has generated exciting results, culminating in fourrecent FDA approvals and the recent availability of commercial productsin the U.S. However, the current personalized cellular product platformis expensive, cumbersome and time-consuming. Specifically, the state ofthe art of gene transfer into T cells for cancer therapy includes avariety of ex vivo approaches that range from viral (e.g. lentiviraltransduction) to non-viral approaches (e.g. electroporation oftransposon plasmids). Patients receive ex vivo manufactured CAR T cells.Among the major clinical issues facing patients and physicians is theduration of the ex vivo manufacturing process, which is at least 17 daysand often much longer. Additional logistical issues that limit access,increase time-to-treatment, and generate costs include apheresisavailability, GMP suites availability in time for manufacturing, andrelease testing of each product, which is thus treated as a new lot.With over 200 CAR T cell trials in progress world-wide and a burgeoningdevelopment pipeline, manufacturing is a critical bottleneck.

SUMMARY OF THE INVENTION

The present invention provides, in part, CAR technology that transformsthe paradigm of CAR T therapy and circumvents ex vivo manufacturingissues. Among other things, the present invention provides methods andcompositions for in vivo CAR gene delivery using an engingeered viralparticle or viral vector system. In particular, the present inventioncontemplates an engineered viral particle or viral vector system thatgenerates cell-based immune responses to specific target cells. Thus,the present invention promises an off-the-shelf CAR drug product moreclosely resembles a traditional pharmaceutical in its ease of use,deliverability and manufacturing. Furthermore, the engineered viralparticle or viral vector system of the present invention may be used todeliver transgenes for treatment of genetic diseases beyond cancer.

Accordingly, in certain aspects, the present disclosure provides anengineered viral particle comprising an engineered envelope harboring amutated fusion protein, a chimeric gag protein, an engineered targetingmoiety for binding to a target cell, wherein the mutated fusion proteindoes not bind to its natural receptor; and a nucleic acid encoding apolypeptide of interest.

In certain exemplary embodiments, the chimeric gag protein is xHIV gagprotein.

In certain exemplary embodiments, the targeting moiety is fused to themutated fusion protein.

In certain exemplary embodiments, the viral particle is a lentiviruspseudotyped with a measles virus (MV) hemagglutinin (HA) protein or anMV fusion (F) protein and the MV-HA protein or the MV-F proteincomprises a mutated binding domain compared to its naturally occurringreceptor.

In certain exemplary embodiments, the targeting moiety is fused to theMV-HA protein or the MV-F protein.

In certain exemplary embodiments, the targeting moiety is a scFv, anantigen binding domain, a DARPIN, a FN3 domain, or any combinationthereof. In certain exemplary embodiments, the targeting moiety isselected from the group consisting of Stem Cell Factor protein (SCF,KIT-ligand, KL, or steel factor) or a moiety that binds to cKit (CD117),CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCRdelta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.

In certain exemplary embodiments, the polypeptide of interest is achimeric antigen receptor (CAR) or a portion thereof. In certainexemplary embodiments, the chimeric antigen receptor comprises anextracellular domain, transmembrane domain, and an intracellularsignaling domain.

In some embodiments, the extracellular domain binds specifically to anantigen expressed at the surface of a cell. In other embodiments, theantigen is a marker expressed by both normal cells and cancer cells,e.g., a lineage marker, e.g., CD19 or CD123 on B cells. In oneembodiment, the antigen is a cell surface molecule that is overexpressedin a cancer cell in comparison to a normal cell. In another embodiment,the antigen is a cell surface molecule that is aberrantly expressed on acancer cell, for example, a molecule that comprises deletions, additionsor mutations in comparison to the molecule expressed on a normal cell.In some embodiments, the antigen will be expressed exclusively on thecell surface of a cancer cell, entirely or as a fragment, and notexpressed on the surface of a normal cell.

In certain exemplary embodiments, the extracellular domain binds to CD8,CCR7, CD20, CD22, CD123, CD38, CD19, BCMA, CD33, or CD79b.

In certain exemplary embodiments, the polypeptide of interest is ahemoglobin beta chain.

In certain exemplary embodiments, the viral particle is a pseudotypedlentiviral vector, an adenovirus, or an adeno-associated virus. Incertain exemplary embodiments, the pseudotyped lentiviral vector ispseudotyped with a paramyxovirus, such as a morbillivirus, such as ameasles virus glycoprotein or a henipavirus such as Nipah virus (NiV)glycoprotein. In certain exemplary embodiments, the pseudotyped vectoris pseudotyped with a F protein or H protein of a morbillivirus. In someembodiments, the pseudotyped vector is pseudotyped with a G protein of aNipah Virus.

In another aspect, the instant disclosure provides a method of in vivogene delivery comprising administering an engineered viral particle to asubject in need of delivery of a protein of interest or a nucleic acidmolecule of interest. In some embodiments, the engineered viral particlecomprises an engineered envelope harboring a mutated fusion protein, achimeric gag protein and an engineered targeting moiety for binding to atarget cell, wherein the mutated fusion protein does not bind to itsnatural receptor; and a nucleic acid encoding a polypeptide of interest.The administration of the engineered viral particle induces an in vivoactivity in the target cell associated with the polypeptide of interest.

In certain exemplary embodiments, the chimeric gag protein is xHIV gagprotein.

In certain exemplary embodiments, the target cell is a T cell, a CD4+ Tcell, a CD8+ T cell, a NK cell, an alpha-beta T cell, a gamma-delta Tcell, a lymphoid progenitor cell, a hematopoietic stem cell, a myeloidcell, a monocyte, a macrophage, a central memory T cell, a naïve T cell,an activated T cell, a regulatory T Cell (Treg), or aT-Cell^(CD8+CCR7+).

In another aspect, the instant disclosure provides a cell comprising apolypeptide of interest encoded for by any of the engineered viralparticles disclosed herein. In certain exemplary embodiments, the cellis a T cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta Tcell, a gamma-delta T cell, a lymphoid progenitor cell, a hematopoieticstem cell, a myeloid cell, a monocyte, a macrophage, a central memory Tcell, a naïve T cells, an activated T cell, or a regulatory T Cell(Treg), or a T-Cell^(CD8+CCR7+).

In another aspect, the instant disclosure provides a method of treatinga disease in a subject. The method comprises administering to thesubject any of the engineered viral particles disclosed herein, whereinthe engineered viral particle expresses the polypeptide of interest in acell. In certain exemplary embodiments, the disease is cancer or sicklecell disease, or genetic disease of the bone marrow or ahemoglobinopathy. In certain exemplary embodiments, the cell is a Tcell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T cell, agamma-delta T cell, a lymphoid progenitor cell, a hematopoietic stemcell, a myeloid cell, a monocyte, a macrophage, a central memory T cell,a naïve T cells, an activated T cell, a regulatory T Cell (Treg), or aT-Cell^(CD8+CCR7+).

In another aspect, the instant disclosure provides a viral vector systemcomprising a first viral particle and a second viral particle. The firstviral particle comprises a chimeric gag protein, a first targetingmoiety that binds to a first target on a cell and a first nucleic acidmolecule that encodes a first portion of a protein or a polypeptide ofinterest. The second viral particle comprises a chimeric gag protein, asecond targeting moiety that binds to a second target on the cell and asecond nucleic acid molecule that encodes a second portion of theprotein or the polypeptide of interest. The first and second portions ofthe protein or the polypeptide are capable of forming a complete proteinor polypeptide of interest inside the cell. In certain exemplaryembodiments, the chimeric gag protein is xHIV gag protein.

In certain exemplary embodiments, the first target and the second targetare different.

In certain exemplary embodiments, the protein or polypeptide of interestis a chimeric antigen receptor. In certain exemplary embodiments, thechimeric antigen receptor comprises an extracellular domain,transmembrane domain, and an intracellular signaling domain. In certainexemplary embodiments, the extracellular domain binds to CD8, CCR7,CD20, CD22, CD123, CD38, CD19, BCMA, CD33, or CD79b.

In certain exemplary embodiments, the protein or polypeptide is ahemoglobin beta chain.

In certain exemplary embodiments, the first targeting moiety and thesecond targeting moiety are each independently selected from the groupconsisting of a protein that binds to the first target, an antigenbinding domain, a DARPIN, and a FN3 domain, or any combination thereof.In certain exemplary embodiments, the first targeting moiety and thesecond targeting moiety are each independently selected from the groupconsisting of Stem Cell Factor protein (SCF, KIT-ligand, KL, or steelfactor) or a moiety that binds to cKit (CD117), CD4, CD8, CD3, CD5, CD6,CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110,CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,and CXCR3. In certain exemplary embodiments, the first targeting moietyis selected from the group consisting of Stem Cell Factor protein (SCF,KIT-ligand, KL, or steel factor) or a moiety that binds to cKit (CD117),CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCRdelta, CD10, CD34, CD110, CD33, CD14, or CD68. In certain exemplaryembodiments, the second targeting moiety binds to CCR7, CD62L, CD25,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3.

In certain exemplary embodiments, the first target and the second targetare each independently selected from the group consisting of cKit(CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCRgamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L,CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3. In certainexemplary embodiments, the first target is cKit (CD117), CD4, CD8, CD3,CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10,CD34, CD110, CD33, CD14, or CD68. In certain exemplary embodiments, thesecond target is CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,or CXCR3.

In certain exemplary embodiments, the first viral vector is apseudotyped viral vector. In certain exemplary embodiments, the secondviral vector is a pseudotyped viral vector.

In certain exemplary embodiments, the first and second viral vector areeach, independently, a pseudotyped lentiviral vector, an adenovirus, oran adeno-associated virus. In certain exemplary embodiments, thepseudotyped lentiviral vector is pseudotyped with a morbillivirus, suchas a measles virus, glycoprotein and/or a Nipah virus glycoprotein. Incertain exemplary embodiments, the first and/or second viral vector ispseudotyped with a F protein or H protein of a morbillivirus.

In certain exemplary embodiments, the cell is a T cell, a CD4+ T cell, aCD8+ T cell, a NK cell, an alpha-beta T cell, a gamma-delta T cell, alymphoid progenitor cell, a hematopoietic stem cell, a myeloid cell, amonocyte, a macrophage, a central memory T cell, a naïve T cell, anactivated T cell, a regulatory T Cell (Treg), or a T-Cell^(CD8+CCR7+).

In certain exemplary embodiments, the protein or polypeptide is achimeric antigen receptor or a hemoglobin beta chain.

In certain exemplary embodiments, the first and second portions of theprotein or polypeptide form a complete protein in the presence of adimerizing agent. In certain exemplary embodiments, the dimerizing agentis rimiducid((1R)-3-(3,4-dimethoxyphenyl)-1-[3-({[2-(2-{3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyloxy]propyl]phenoxy}acetamido)ethyl]carbamoyl}methoxy)phenyl]propyl(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate(AP1903)), erythropoietin or the dimerizing agent can be a soluble drugor hormone that naturally binds homodimers or heterodimers such aserythropoietin or thrombopoietin, or rapamycin.

In certain exemplary embodiments, the first and second portions of theprotein or polypeptide can bind together through the excision of anintein domain.

In certain exemplary embodiments, the first portion of the proteincomprises an extracellular domain of a chimeric antigen receptor and thesecond portion of the protein comprises the transmembrane domain, and anintracellular signaling domain.

In another aspect, the instant disclosure provides a cell comprising anyof the viral vector systems disclosed herein. In certain exemplaryembodiments, the cell is a T cell, a CD4+ T cell, a CD8+ T cell, a NKcell, an alpha-beta T cell, a gamma-delta T cell, a lymphoid progenitorcell, a hematopoietic stem cell, a myeloid cell, a monocyte, amacrophage, a central memory T cell, a naïve T cells, an activated Tcell, or a regulatory T Cell (Treg), or a T-Cell^(CD8+CCR7+).

In another aspect, the instant disclosure provides a method of in vivogene delivery comprising administering to a subject in need thereof afirst viral particle and a second viral particle. The first viralparticle comprises a chimeric gag protein, a first targeting moiety thatbinds to a first target on a cell and a first nucleic acid molecule thatencodes a first portion of a protein or a polypeptide of interest. Thesecond viral particle comprises a chimeric gag protein, a secondtargeting moiety that binds to a second target on the cell and a secondnucleic acid molecule that encodes a second portion of the protein orthe polypeptide of interest. The first and second portions of theprotein or the polypeptide are capable of forming a complete protein orpolypeptide of interest inside the cell. In certain exemplaryembodiments, the chimeric gag protein is xHIV gag protein.

In certain exemplary embodiments, the method further comprisesadministering a dimerizing agent to form the protein or polypeptide inthe subject.

In certain exemplary embodiments, the first portion and the secondportion comprise an intein domain and the intein domain is excised toconjugate the first and second portion to form the protein orpolypeptide.

In certain exemplary embodiments, the cell is a T cell, a CD4+ T cell, aCD8+ T cell, a NK cell, an alpha-beta T cell, a gamma-delta T cell, alymphoid progenitor cell, a hematopoietic stem cell, a myeloid cell, amonocyte, a macrophage, a central memory T cell, a naïve T cells, anactivated T cell, a regulatory T Cell (Treg), or a CD8+ naïve/centralmemory cell (CD8+CCR7+) and CD4+ naïve/central memory cell (CD4+CCR7+).

In certain exemplary embodiments, the protein or polypeptide is achimeric antigen receptor. In certain exemplary embodiments, the proteinor polypeptide is a hemoglobin beta chain.

In certain exemplary embodiments, the first viral vector is apseudotyped viral vector. In certain exemplary embodiments, the secondviral vector is a pseudotyped viral vector. In certain exemplaryembodiments, the first and second viral vector are each, independently,a pseudotyped lentiviral vector, an adenovirus, or an adeno-associatedvirus. In certain exemplary embodiments, the pseudotyped lentiviralvector is pseudotyped with a morbillivirus, such as a measles virusglycoprotein and/or a Nipah virus glycoprotein. In certain exemplaryembodiments, the first and/or second viral vector is pseudotyped with aF protein or H protein of a morbillivirus.

In certain exemplary embodiments, an engineered viral particle isprovided comprising an engineered envelope comprising a polypeptidehaving the amino acid sequence of SEQ ID NO: 9; a heterologouspolypeptide targeting moiety for binding to a target cell; and a nucleicacid molecule encoding a heterologous polypeptide of interest.

In another aspect, the instant disclosure provides a method of treatinga disease in a subject. The method comprises administering to thesubject a first viral particle and a second viral particle. The firstviral particle comprises a chimeric gag protein, a first targetingmoiety that binds to a first target on a cell and a first nucleic acidmolecule that encodes a first portion of a protein or a polypeptide ofinterest. The second viral particle comprises a chimeric gag protein, asecond targeting moiety that binds to a second target on the cell and asecond nucleic acid molecule that encodes a second portion of theprotein or the polypeptide of interest. The first and second portions ofthe protein or the polypeptide are capable of forming a complete proteinor polypeptide of interest inside the cell.

In some embodiments, the disease is a cancer or a genetic diseaseincluding, but not limited to, e.g., bone marrow disease,hemoglobinopathy, and sickle cell disease.

In certain exemplary embodiments, the disease is cancer or sickle celldisease.

In certain exemplary embodiments, the method further comprisesadministering a dimerizing agent to form the protein or polypeptide inthe cell.

In certain exemplary embodiments, the first portion and the secondportion comprise an intein domain and the intein domain is excised toconjugate the first and second portion to form the protein orpolypeptide.

In certain exemplary embodiments, the cell is a T cell, a CD4+ T cell, aCD8+ T cell, a NK cell, an alpha-beta T cell, a gamma-delta T cell, alymphoid progenitor cell, a hematopoietic stem cell, a myeloid cell, amonocyte, a macrophage, a central memory T cell, a naïve T cells, anactivated T cell, a regulatory T Cell (Treg), or a T-Cell^(CD8+CCR7+).

In certain exemplary embodiments, the protein or polypeptide is achimeric antigen receptor. In certain exemplary embodiments, the proteinor polypeptide is a hemoglobin beta chain.

In certain exemplary embodiments, the first viral vector is apseudotyped viral vector. In certain exemplary embodiments, the secondviral vector is a pseudotyped viral vector. In certain exemplaryembodiments, the first and second viral vector are each, independently,a pseudotyped lentiviral vector, an adenovirus, or an adeno-associatedvirus. In certain exemplary embodiments, the pseudotyped lentiviralvector is pseudotyped with a morbillivirus, such as a measles virus,glycoprotein and/or a Nipah virus glycoprotein. In certain exemplaryembodiments, the first and/or second viral vector is pseudotyped with aF protein or H protein of a morbillivirus.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the embodimentsprovided herein will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings.

FIGS. 1A-1B are schematics illustrating measles virus (MV)-pseudotypedlentiviral vectors and viral vector systems. FIG. 1A illustrates aMV-pseudotyped lentiviral vector system comprising an HIV gag-polsequence. FIG. 1B is a schematic illustrating a non-limiting embodimentof a MV-pseudotyped lentiviral vector system, wherein the HIV gagsequence is replaced with a sequence encoding xHIV (SEQ ID NO: 5), achimeric gag protein from SIV and HIV.

FIGS. 2A-2D depict results from experiments wherein activated PBMCs weretransduced with gag-pol NGFR MV (FIG. 2A and FIG. 2C) or xHIV NGFR MV(FIG. 2B and FIG. 2D). Results from day 4 are shown.

FIGS. 3A-3D depict results from experiments wherein activated PBMCs weretransduced with gag-pol GFP MV (FIG. 3A and FIG. 3C) or xHIV GFP MV(FIG. 3B and FIG. 3D). Results from day 4 are shown.

FIGS. 4A-4D depict results from experiments wherein activated PBMCs weretransduced with gag-pol CAR-19 MV (FIG. 4A and FIG. 4C) or xHIV CAR-19MV (FIG. 4B and FIG. 4D). Results from day 4 are shown.

FIGS. 5A-5D depict results from experiments wherein activated PBMCs weretransduced with gag-pol NGFR MV (FIG. 5A and FIG. 5C) or xHIV NGFR MV(FIG. 5B and FIG. 5D). Results from day 12 are shown.

FIGS. 6A-6D depict results from experiments wherein activated PBMCs weretransduced with gag-pol GFP MV (FIG. 6A and FIG. 6C) or xHIV GFP MV(FIG. 6B and FIG. 6D). Results from day 12 are shown.

FIGS. 7A-7D depict results from experiments wherein activated PBMCs weretransduced with gag-pol CAR-19 MV (FIG. 7A and FIG. 7C) or xHIV CAR-19MV (FIG. 7B and FIG. 7D). Results from day 12 are shown.

FIGS. 8A-8B is a schematic illustrating a split vector application inaccordance with some embodiments of the present disclosure. (A) Asdescribed in Example 2, EpoR signaling (e.g., EpoR 4-1BB-CD3 zeta (BBz)endodomain encoded by transgene 1 from lentiviral vector 1 (LV 1)) andEpoR binding (e.g., the CAR-EpoR exodomain encoded by transgene 2 fromlentiviral vector 2 (LV 2)) components are individually expressed in acell using different lentiviral vectors for each. (B) Dimerizationdomains of EpoR dimerize to provide signaling activity in the presenceof the dimerizing agent EPO.

FIG. 9 depicts results from experiments of expression of CAR-EpoRexodomain described in Example 2.

FIG. 10 depicts results from experiments of expression of EpoR BBzendodomain described in Example 2.

FIG. 11 is a schematic illustrating another embodiment of the splitvector application where only CD8+CCR7+ cells contain both the exo- andendo-domains, which can be activated via the CAR and with administrationof EPO as described in Example 2.

FIG. 12 depicts results from experiments of specific transduction ofhuman CD4 cells in blood of humanized mice with CD4-targeting MV system,GFP reporter, 7 days after IV administration of vector.

FIG. 13 depicts results from experiments of specific transduction ofhuman CD4 cells in liver of humanized mice with CD4-targeting MV system,GFP reporter, 7 days after IV administration of vector. VSVG controlshows non-specific transduction of liver Kupffer cells.

FIG. 14 depicts results from experiments of specific transduction ofhuman CD4 cells in peritoneum of humanized mice with CD4-targeting MVsystem, NGFR reporter, 7 days after IV administration of vector.Triplicate mice are shown.

FIG. 15 depicts results from experiments of specific transduction ofresting (non-activated) rhesus macaque CD4 cells in vitro. NGFRreporter. VSVG control shows no transduction since the cells are notactivated. MV and NV systems show equivalently high and specifictransduction.

FIG. 16 depicts results from experiments of human T cells stimulated,transduced with MV-pseudotyped, anti-CD8 DARPIN redirected vector madeusing xHIV gag and activated with IL7 and IL15. Flow cytometry on day 11shows efficient and specific transgene expression. Individual FACS plotsshow expression at 3-fold dilutions of viral vector.

FIG. 17 depicts results from experiments of human T cells stimulated,transduced with MV-pseudotyped, anti-CD8 DARPIN redirected vector madeusing xHIV gag and activated with IL7 and IL15. Flow cytometry on day 11shows efficient and specific transgene expression. Exemplary FACS plotfrom slide 1 is shown as representation. Transduction efficiency is20.7/20.7+12.2=63%.

FIG. 18 depicts results from experiments of degranulation in response totwo different CD19+CD20+ lymphoma cell lines with anti-CD20 or anti-CD19BBz CAR20 (VSVG) or antiCD20 or anti-CD19 KIR MV or NiV systems.

FIG. 19 depicts results from experiments of killing of CD20+ lymphomacell line with anti-CD20 BBz CAR20 (VSVG) or antiCD20 KIR MV. KIR20system is slower to respond (kill) than standard BBz-costimulated CARsystem.

FIG. 20 depicts results from experiments wherein incorporation of anIL15/IL15R expression cassette drives proliferation of transduced cells.10-fold higher expansion of cells transduced to express the cytokineresponse cassette compared to controls.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used hereinhave the meanings that are commonly understood by those of ordinaryskill in the art. In the event of any latent ambiguity, definitionsprovided herein take precedent over any dictionary or extrinsicdefinition. Unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein is well-knownand commonly used in the art. The methods and techniques provided hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The nomenclatures usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

That the disclosure may be more readily understood, select terms aredefined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

“Activation,” as used herein in reference to a T cell, refers to thestate of a T cell that has been sufficiently stimulated to inducedetectable cellular proliferation. Activation can also be associatedwith induced cytokine production, and detectable effector functions. Theterm “activated T cells” refers to, among other things, T cells that areundergoing cell division.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including virtually all proteins or peptides, can serveas an antigen. The term “antigen” can also refer to a molecule that anantibody or antibody-like molecule can bind to or is recognized by theantibody or antibody-like molecule.

The term “antibody molecule,” “antibody” or antigen binding domain, asthat term is used herein, refers to a polypeptide, e.g., animmunoglobulin chain or fragment thereof, comprising at least onefunctional immunoglobulin variable domain sequence. An antibody moleculeencompasses antibodies (e.g., full-length antibodies) and antibodyfragments. In some embodiments, an antibody molecule comprises anantigen binding or functional fragment of a full-length antibody, or afull-length immunoglobulin chain. For example, a full-length antibody isan immunoglobulin (Ig) molecule (e.g., an IgG antibody) that isnaturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes. In embodiments, an antibody molecule refersto an immunologically active, antigen binding portion of animmunoglobulin molecule, such as an antibody fragment. An antibodyfragment, e.g., functional fragment, comprises a portion of an antibody,e.g., Fab, Fab′, F(ab′)2, F(ab)2, variable fragment (Fv), domainantibody (dAb), or single chain variable fragment (scFv). A functionalantibody fragment binds to the same antigen as that recognized by theintact (e.g., full-length) antibody. The terms “antibody fragment” or“functional fragment” also include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains or recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”). In some embodiments, anantibody fragment does not include portions of antibodies withoutantigen binding activity, such as Fc fragments or single amino acidresidues. Exemplary antibody molecules include full-length antibodiesand antibody fragments, e.g., dAb (domain antibody), single chain, Fab,Fab′, and F(ab′)2 fragments, and single chain variable fragments(scFvs).

The term “antibody molecule” also encompasses whole or antigen bindingfragments of domain, or single domain, antibodies, which can also bereferred to as “sdAb” or “VHH.” Domain antibodies comprise either VH orVL that can act as stand-alone, antibody fragments. Additionally, domainantibodies include heavy-chain-only antibodies (HCAbs). Domainantibodies also include a CH2 domain of an IgG as the base scaffold intowhich CDR loops are grafted. It can also be generally defined as apolypeptide or protein comprising an amino acid sequence that iscomprised of four framework regions interrupted by three complementaritydetermining regions. This is represented asFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. sdAbs can be produced in camelids suchas llamas, but can also be synthetically generated using techniques thatare well known in the art. The numbering of the amino acid residues of asdAb or polypeptide is according to the general numbering for VH domainsgiven by Kabat et al. (“Sequence of proteins of immunological interest,”US Public Health Services, NIH Bethesda, Md., Publication No. 91, whichis hereby incorporated by reference). According to this numbering, FR1of a sdAb comprises the amino acid residues at positions 1-30, CDR1 of asdAb comprises the amino acid residues at positions 31-36, FR2 of a sdAbcomprises the amino acids at positions 36-49, CDR2 of a sdAb comprisesthe amino acid residues at positions 50-65, FR3 of a sdAb comprises theamino acid residues at positions 66-94, CDR3 of a sdAb comprises theamino acid residues at positions 95-102, and FR4 of a sdAb comprises theamino acid residues at positions 103-113. Domain antibodies are alsodescribed in WO2004041862 and WO2016065323, each of which is herebyincorporated by reference. The domain antibodies can be a targetingmoiety as described herein.

Antibody molecules can be monospecific (e.g., monovalent or bivalent),bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, orhexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, orhexavalent), or with higher orders of specificity (e.g, tetraspecific)and/or higher orders of valency beyond hexavalency. An antibody moleculecan comprise a functional fragment of a light chain variable region anda functional fragment of a heavy chain variable region, or heavy andlight chains may be fused together into a single polypeptide.

Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample. Such a biological sample can include, but is notlimited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

As used herein, the term “autologous” is meant to refer to any material,such as a cell, derived from a subject to which it is later to bere-introduced into the same subject.

As used herein, the term “allogeneic” is meant to refer to material,such as a cell, derived from one subject that is later introduced into adifferent subject.

A “co-stimulatory molecule” or “co-stimulatory receptor” refers to thecognate binding partner on a T cell that specifically binds with aco-stimulatory ligand, thereby mediating a co-stimulatory response bythe T cell, such as, but not limited to, proliferation, activation,differentiation, and the like. Co-stimulatory molecules include, but arenot limited to CD27, CD28, CD40, or 4-1BB.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an artificial antigen presentingcell or “aAPC”, dendritic cell, B cell, and the like) that specificallybinds a cognate co-stimulatory receptor on a T cell, thereby providing asignal which, in addition to the primary signal provided by, forinstance, binding of a TCR/CD3 complex to an WIC molecule loaded withpeptide, mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited toan amount that when administered to a mammal, causes a detectable levelof immune cell activation compared to the immune cell activationdetected in the absence of the composition. The immune response can bereadily assessed by a plethora of art-recognized methods. The skilledartisan would understand that the amount of the composition administeredherein varies and can be readily determined based on a number of factorssuch as the disease or condition being treated, the age and health andphysical condition of the mammal being treated, the severity of thedisease, the particular compound being administered, and the like.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly about 10 amino acids and/or sugars in size. In someembodiments, the epitope is about 4-18 amino acids, about 5-16 aminoacids, about 6-14 amino acids, about 7-12, or about 8-10 amino acids.One skilled in the art understands that generally the overallthree-dimensional structure, rather than the specific linear sequence ofthe molecule, is the main criterion of antigenic specificity and,therefore, distinguishes one epitope from another. Based on the presentdisclosure, a peptide can be an epitope.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., Sendai viruses, lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

As used herein, the phrase “ex vivo” in reference to a cell beingtransduced, tranfected or transformed ex vivo, refers to a cell beingtransduced, tranfected or transformed outside of the subject, that iswith the cells being removed from the subject before such cells aretransduced, tranfected or transformed.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two nucleic acid oramino acid molecules, such as, between two polynucleotide or polypeptidemolecules. When two amino acid sequences have the same residues at thesame positions; e.g., if a position in each of two polypeptide moleculesis occupied by an Arginine, then they are identical at that position.The identity or extent to which two amino acid or two nucleic acidsequences have the same residues at the same positions in an alignmentis often expressed as a percentage. The identity between two amino acidor two nucleic acid sequences is a direct function of the number ofmatching or identical positions; e.g., if half of the positions in twosequences are identical, the two sequences are 50% identical; if 90% ofthe positions (e.g., 9 of 10), are matched or identical, the two aminoacids sequences are 90% identical.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e³ and e¹⁰⁰ indicating a closely related sequence.

To the extent the invention includes composition comprising variousproteins, these proteins may, in some instances, comprise amino acidsequences that have sequence identity to the amino acid sequencesdisclosed herein. Therefore, in certain embodiments, depending on theparticular sequence, the degree of sequence identity is preferablygreater than 50% (e.g. 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) to the SEQ ID NOs disclosed herein.These proteins may include homologs, orthologues, allelic variants andfunctional mutants. Typically, 50% identity or more between twopolypeptide sequences is considered to be an indication of functionalequivalence. Identity between polypeptides is preferably determined bythe Smith-Waterman homology search algorithm as implemented in theMPSRCH program (Oxford Molecular), using an affine gap search withparameters gap open penalty—12 and gap extension penalty=1.

These proteins may, compared to the disclosed proteins, include one ormore (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acidreplacements i.e. replacements of one amino acid with another which hasa related side chain. Genetically-encoded amino acids are generallydivided into four families: (1) acidic i.e. aspartate, glutamate; (2)basic i.e. lysine, arginine, histidine; (3) non polar i.e. alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Ingeneral, Substitution of single amino acids within these families doesnot have a major effect on the biological activity. The proteins mayhave one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single aminoacid deletions relative to the disclosed protein sequences. The proteinsmay also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to thedisclosed protein sequences.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen. In some embodiments, theimmune response can be against a tumor cell expressing the antigen. Insome embodiments, the immune response is facilitated by a T cellexpressing a chimeric antigen receptor, such as those, but not limitedto, those provided herein.

The term “immunosuppressive” is used herein to refer to reducing overallimmune response.

As used herein, the phrase “in vivo” in reference to a cell beingtransduced, tranfected or transformed in vivo, refers to a cell beingtransduced, tranfected or transformed in the subject without the cellsbeing removed from the subject before such cells are transduced,tranfected or transformed.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily that is able to infect non-dividing cells. Lentiviruses candeliver a significant amount of genetic information into the DNA of thehost cell, so they are one of the most efficient methods of a genedelivery vector. HIV, SIV, and FIV are all examples of lentiviruses.Vectors derived from lentiviruses offer the ability to achieve genetransfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell as provided herein. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids or the expression of heterologous proteins.

By the term “modulating,” as used herein, is meant mediating an increaseor decrease in the level of a response in a subject compared with thelevel of a response in the subject in the absence of a treatment orcompound, and/or compared with the level of a response in an otherwiseidentical but untreated subject. The term encompasses perturbing and/oraffecting a native signal or response thereby mediating a beneficialtherapeutic response in a subject, such as, a human.

As used herein, the following abbreviations for the commonly occurringnucleic acid bases are used: “A” refers to adenosine, “C” refers tocytosine, “G” refers to guanosine, “T” refers to thymidine, and “U”refers to uridine.

The “Nipah virus” (NiV) is member of the family Paramyxoviridae, genusHenipavirus. Nipah virus is an enveloped virus with negative-strandedpolarity and a non-segmented RNA genome consisting of helicalnucleocapsids. Two strains of Nipah virus include, but are not limitedto, the Malaysian (MY) and the Bangladesh (BD) strains.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “oligonucleotide” typically refers to short polynucleotides. Itwill be understood that when a nucleotide sequence is represented by aDNA sequence (i.e., A, T, C, G), this also provides the correspondingRNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

“Parenteral” administration of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, the terms “nucleic acids” and “polynucleotides” as used herein areinterchangeable. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any methodsavailable in the art, including, without limitation, recombinantmethods, i.e., the cloning of nucleic acid sequences from a recombinantlibrary or a cell genome, using cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of a pluralityof amino acid residues covalently linked by peptide bonds. As usedherein, the term refers to both short chains, which also commonly arereferred to in the art as peptides, oligopeptides and oligomers, forexample, and to longer chains, which generally are referred to in theart as proteins, of which there are many types. “Polypeptides” include,for example, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “pseudotyped” or “pseudotyped viral particle”, as used herein,refers to a viral particle bearing envelope glycoproteins derived fromother viruses having envelopes or a viral vector encoding envelopeglycoproteins from a virus that is different from the parental virus.The host range of the vector particles can thus be expanded or altereddepending on the type of cell surface receptor used by the glycoprotein.For example, a HIV lentiviral vector can have the HIV envelopeglycoprotein be replaced with the VSV envelope glycoprotein. This isjust one non-limiting example and other envelop glycoproteins can beused, such as the envelope glycoprotein of the Nipah virus. Therefore,in some embodiments, the viral particle is encoded by a lentivirus thatencodes the Nipah viral envelope glycoprotein. In some embodiments, theNipah viral envelope glycoprotein is glycoprotein F. In someembodiments, the Nipah viral envelope glycoprotein is glycoprotein G. Insome embodiments, the pseudotyped viral vector encodes both the Nipahviral glycoprotein F and glycoprotein G. In some embodiments, thepseudotyped viral particle expresses one or both of the Nipah viralglycoprotein F and glycoprotein G.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” includes living organisms, including those in whichan immune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, non-human primates, feline and murinemammals. In some embodiments, the subject is human.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (α) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into a cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny. In some embodiments, the transfection, transformation, ortransduction is performed or occurs in vivo.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

As used herein, the term “variant” when used in conjunction to an aminoacid sequence refers to a sequence that is at least, or about, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thereference sequence. In some embodiments, the variant comprises 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, thesubstitution is a conservative substitution.

A “vector” is a composition of matter which comprises an isolatednucleic acid encoding a protein or a peptide. Numerous vectors are knownin the art including, but not limited to, linear polynucleotides,plasmids, DNA, and RNA. Examples of viral vectors include, but are notlimited to, Sendai viral vectors, adenoviral vectors, adeno-associatedvirus vectors, retroviral vectors, lentiviral vectors, and the like.

A “carrier” or “delivery vehicle” includes viral particles, viruses,polylysine compounds, and liposomes, which facilitate transfer ofnucleic acid into cells. A carrier or delivery vehicle can also be usedto deliver a protein or peptide to a cell.

As used herein, xHIV and xHIV (i.e., Greek symbol χ(chi)HIV) are usedsynonymously so as to avoid any confusion derived from electronic orother imaging of this specification.

Ranges: throughout this disclosure, various aspects of the embodimentscan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This appliesregardless of the breadth of the range. Unless otherwise explicitlystated to the contrary, a range that is disclosed also includes theendpoints of the range.

DETAILED DESCRIPTION

The present invention provides, among other things, compositions andmethods for transducing cells (e.g. T cells) in vivo. In someembodiments, these compositions and methods are performed or usedwithout culturing or transducing the cells ex-vivo. In some embodiments,a patient with a malignancy requiring CAR T cell therapy can be treatedwith an off-the-shelf composition that transduces their T cells in vivo,which generates CAR T cells in situ. These methods and compositions,such as a gene transfer vector (e.g. lentiviral vector) in viralparticles, as opposed to ex-vivo transduced cells, can be used, forexample, to turn the patient's own lymphoid organs into a bioreactor toproduce the CAR T cells. In some embodiments, vectors that can beutilized are those that transduce CD4+CD8+ T cells with lentiviruses,such as a CD4-specific lentivirus and/or a CD8-specific lentivirus suchthat only the targeted cells that express both CD4 and CD8 express thetransgene of interest based on the combination of the expressionproducts being expressed from the plurality of lentiviruses being usedto transduce the cells in vivo.

It is to be understood that the methods described in this disclosure arenot limited to particular methods and experimental conditions disclosedherein as such methods and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Furthermore, the experiments described herein, unless otherwiseindicated, use conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008),including all supplements, Molecular Cloning: A Laboratory Manual(Fourth Edition) by MR Green and J. Sambrook and Harlow et al.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor (2013, 2nd edition).

Engineered Viral Particles

The present invention provides, among other things, compositions thatcan be used, for example, to selectively transduce a cell expressingspecific cell surface markers using viral particles. In someembodiments, the viral particles are used to transduce the cells invivo. In some embodiments, the viral particles are used to transduce thecells ex vivo. By utilizing viral particles to transduce the same celltype, the viral particles can increase specificity, improve safety bylimiting expression of the entire molecule of interest to the specificcell type, and in certain embodiments, provide an additional layer ofcontrol by utilizing dimerization domains that require administration ofan exogenous agent so that the molecule of interest will form from thefirst and second portions expressed by the viral particle.

Viral Vector System

The present invention also provides, among other things, compositionsthat can be used, for example, to selectively transduce a cellexpressing specific cell surface markers in vivo or ex vivo using aplurality of viral particles. By utilizing a plurality of viralparticles to transduce the same cell type, the viral particles canincrease specificity, improve safety by limiting expression of theentire molecule of interest to the specific cell type, and in certainembodiments, provide an additional layer of control by utilizingdimerization domains that require administration of an exogenous agentso that the molecule of interest will form from the first and secondportions expressed by the plurality of vectors.

Protein or Polypeptide of Interest

In some embodiments, the viral particle encodes a polypeptide ofinterest, which can also be referred to as a molecule of interest. Insome embodiments, one viral particle provides a viral vector encoding afirst portion of a molecule of interest, and another viral particleprovides a second viral vector encoding a second portion of a moleculeof interest. As provided herein, in some embodiments, the molecule ofinterest is a chimeric antigen receptor.

In some embodiments, the viral particle delivers a nucleic acid moleculeof interest. In some embodiments, the nucleic acid molecule of interestencodes the polypeptide of interest.

In some embodiments, a composition comprising an engineered viralparticle comprising an engineered envelope harboring a mutated fusionprotein and an engineered targeting moiety for binding to a target cell,wherein the mutated fusion protein does not bind to its naturalreceptor; and a nucleic acid encoding a polypeptide of interest isprovided.

In some embodiments, the gag protein is not chimeric gag and iswild-type gag.

In some embodiments, a composition comprising an engineered viralparticle comprising an engineered envelope harboring a mutated fusionprotein, a chimeric gag protein, and an engineered targeting moiety forbinding to a target cell, wherein the mutated fusion protein does notbind to its natural receptor; and a nucleic acid encoding a polypeptideof interest is provided.

In some embodiments, a composition comprising a first viral particle anda second viral particle are provided. In some embodiments, the firstviral particle comprises a chimeric gag protein, a first targetingmoiety that binds to a first target on a cell, and a nucleic acidmolecule that encodes a first portion of a protein or polypeptide. Insome embodiments, the second viral particle comprises a chimeric gagprotein, a second targeting moiety that binds to a second target on thecell, and a nucleic acid molecule that encodes a second portion of theprotein or the polypeptide. Once expressed in the cell, the first andsecond portions of the protein or the polypeptide can bind together orinteract with one another to form a complete or functional protein orpolypeptide.

In certain embodiments, the chimeric gag protein is xHIV gag protein.The chimeric gag protein xHIV is described in Uchida et al. J. Virol,October 2009, p. 9854-9862, contents of which are incorporated byreference herein.

In certain embodiments, the chimeric gag protein, as described herein,comprises SIV and HIV sequences. In certain embodiments, the chimericgag protein is referred to as xHIV gag protein. In certain embodiments,the xHIV comprises a HIV long-terminal repeat (LTR), a HIV gag protein(gag), a SIV element, a HIV Pol protein (Pol), and a HIV Envelope (Env)protein. In some embodiments, gag, Pol, and/or Env protein is apolyprotein. In certain embodiments, the gag protein, the SIV element,and the Pol protein of the xHIV are encoded by the nucleic acid sequenceas set forth in SEQ ID NO: 5.

(5′-3′ xHIV gag-pol nt, SEQ ID NO: 5)atgggtgcgagagcgtcggtattaagcgggggagaattagataaatgggaaaaaattcggttaaggccagggggaaagaaacaatataaactaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggccttttagagacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacaatagcagtcctctattgtgtgcatcaaaggatagatgtaaaagacactaaggaagccttagataagatagaggaagaacaaaacaaaagtaagaaaaaggcacagcaagcagcagctgacacaggaaacaacagccaggtcagccaaaattacccagtacaacaaataggtggtaactatgtccacctgccattaagcccgagaacattaaatgcctgggtaaaattgatagaggaaaagaaatttggagcagaagtagtgccaggatttcaggcactgtcagaaggttgcaccccctatgacattaatcagatgttaaattgtgtgggagaccatcaagcggctatgcagattatcagagatattataaacgaggaggttgcagattgggacttgcagcacccacaaccagctccacaacaaggacaacttagggagccgtcaggatcagatattgcaggaacaactagttcagtagatgaacaaatccagtggatgtacagacaacagaaccccataccagtaggcaacatttacaggagatggatccaactggggttgcaaaaatgtgtcagaatgtataacccaacaaacattctagatgtaaaacaagggccaaaagagccatttcagagctatgtagacaggttctacaaaagtttaagagcagaacagacagatgcagcagtaaagaattggatgactcaaacactgctgattcaaaatgctaacccagattgcaagctagtgctgaaggggttgggaccaggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccggccataaagcaagagttttggctgaagcaatgagccaagtaacaaatccagctaccataatgatacagaaaggcaattttaggaaccaaagaaagactgttaagtgtttcaattgtggcaaagaagggcacatagccaaaaattgcagggcccctaggaaaaagggctgttggaaatgtggaaaggaaggacaccaaatgaaagattgtactgagagacaggctaattttttagggaagatctggccttcccacaagggaaggccagggaattttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtttggggaagagacaacaactccctctcagaagcaggagccgatagacaaggaactgtatcctttagcttccctcagatcactctttggcagcgacccctcgtcacaataaagataggggggcaattaaaggaagctctattagatacaggagcagatgatacagtattagaagaaatgaatttgccaggaagatggaaaccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgatcagatactcatagaaatctgcggacataaagctataggtacagtattagtaggacctacacctgtcaacataattggaagaaatctgttgactcagattggctgcactttaaattttcccattagtcctattgagactgtaccagtaaaattaaagccaggaatggatggcccaaaagttaaacaatggccattgacagaagaaaaaataaaagcattagtagaaatttgtacagaaatggaaaaggaaggaaaaatttcaaaaattgggcctgaaaatccatacaatactccagtatttgccataaagaaaaaagacagtactaaatggagaaaattagtagatttcagagaacttaataagagaactcaagatttctgggaagttcaattaggaataccacatcctgcagggttaaaacagaaaaaatcagtaacagtactggatgtgggcgatgcatatttttcagttcccttagataaagacttcaggaagtatactgcatttaccatacctagtataaacaatgagacaccagggattagatatcagtacaatgtgcttccacagggatggaaaggatcaccagcaatattccagtgtagcatgacaaaaatcttagagccttttagaaaacaaaatccagacatagtcatctatcaatacatggatgatttgtatgtaggatctgacttagaaatagggcagcatagaacaaaaatagaggaactgagacaacatctgttgaggtggggatttaccacaccagacaaaaaacatcagaaagaacctccattcctttggatgggttatgaactccatcctgataaatggacagtacagcctatagtgctgccagaaaaggacagctggactgtcaatgacatacagaaattagtgggaaaattgaattgggcaagtcagatttatgcagggattaaagtaaggcaattatgtaaacttcttaggggaaccaaagcactaacagaagtagtaccactaacagaagaagcagagctagaactggcagaaaacagggagattctaaaagaaccggtacatggagtgtattatgacccatcaaaagacttaatagcagaaatacagaagcaggggcaaggccaatggacatatcaaatttatcaagagccatttaaaaatctgaaaacaggaaagtatgcaagaatgaagggtgcccacactaatgatgtgaaacaattaacagaggcagtacaaaaaatagccacagaaagcatagtaatatggggaaagactcctaaatttaaattacccatacaaaaggaaacatgggaagcatggtggacagagtattggcaagccacctggattcctgagtgggagtttgtcaatacccctcccttagtgaagttatggtaccagttagagaaagaacccataataggagcagaaactttctatgtagatggggcagccaatagggaaactaaattaggaaaagcaggatatgtaactgacagaggaagacaaaaagttgtccccctaacggacacaacaaatcagaagactgagttacaagcaattcatctagctttgcaggattcgggattagaagtaaacatagtgacagactcacaatatgcattgggaatcattcaagcacaaccagataagagtgaatcagagttagtcagtcaaataatagagcagttaataaaaaaggaaaaagtctacctggcatgggtaccagcacacaaaggaattggaggaaatgaacaagtagataaattggtcagtgctggaatcaggaaagtactatttttagatggaatagataaggcccaagaagaacatgagaaatatcacagtaattggagagcaatggctagtgattttaacctaccacctgtagtagcaaaagaaatagtagccagctgtgataaatgtcagctaaaaggggaagccatgcatggacaagtagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggaaaggaccagcaaagctcctctggaaaggtgaaggggcagtagtaatacaagataatagtgacataaaagtagtgccaagaagaaaagcaaagatcatcagggattatggaaaacagatggcaggtgatgattgtgtggcaagtagacaggatgaggattaa.

In certain embodiments, the gag protein is encoded by the nucleic acidsequence as set forth in SEQ ID NO: 6.

(5′-3′ xHIV gag protein nt, SEQ ID NO: 6)atgggtgcgagagcgtcggtattaagcgggggagaattagataaatgggaaaaaattcggttaaggccagggggaaagaaacaatataaactaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggccttttagagacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacaatagcagtcctctattgtgtgcatcaaaggatagatgtaaaagacactaaggaagccttagataagatagaggaagaacaaaacaaaagtaagaaaaaggcacagcaagcagcagctgacacaggaaacaacagccaggtcagccaaaattacccagtacaacaaataggtggtaactatgtccacctgccattaagcccgagaacattaaatgcctgggtaaaattgatagaggaaaagaaatttggagcagaagtagtgccaggatttcaggcactgtcagaaggttgcaccccctatgacattaatcagatgttaaattgtgtgggagaccatcaagcggctatgcagattatcagagatattataaacgaggaggttgcagattgggacttgcagcacccacaaccagctccacaacaaggacaacttagggagccgtcaggatcagatattgcaggaacaactagttcagtagatgaacaaatccagtggatgtacagacaacagaaccccataccagtaggcaacatttacaggagatggatccaactggggttgcaaaaatgtgtcagaatgtataacccaacaaacattctagatgtaaaacaagggccaaaagagccatttcagagctatgtagacaggttctacaaaagtttaagagcagaacagacagatgcagcagtaaagaattggatgactcaaacactgctgattcaaaatgctaacccagattgcaagctagtgctgaaggggttgggaccaggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccggccataaagcaagagttttggctgaagcaatgagccaagtaacaaatccagctaccataatgatacagaaaggcaattttaggaaccaaagaaagactgttaagtgtttcaattgtggcaaagaagggcacatagccaaaaattgcagggcccctaggaaaaagggctgttggaaatgtggaaaggaaggacaccaaatgaaagattgtactgagagacaggctaattttttagggaagatctggccttcccacaagggaaggccagggaattttcttcagagcagaccagagccaacagccccaccagaagagagatcaggtttggggaagagacaacaactccctctcagaagcaggagccgatagacaaggaactgtatcctttagcttccctcagatcactctttggcagcgacccctcgtcacaataa.

In certain embodiment, the SIV element is encoded by the nucleic acidsequence as set forth in SEQ ID NO: 7. In certain embodiments, thenucleic acid sequence encoding the SIV element is located within thenucleic acid sequence encoding the gag protein as set forth in SEQ IDNO: 6.

(5′-3′ xHIV SIV element nt, SEQ ID NO: 7)aaaattacccagtacaacaaataggtggtaactatgtccacctgccattaagcccgagaacattaaatgcctgggtaaaattgatagaggaaaagaaatttggagcagaagtagtgccaggatttcaggcactgtcagaaggttgcaccccctatgacattaatcagatgttaaattgtgtgggagaccatcaagcggctatgcagattatcagagatattataaacgaggaggttgcagattgggacttgcagcacccacaaccagctccacaacaaggacaacttagggagccgtcaggatcagatattgcaggaacaactagttcagtagatgaacaaatccagtggatgtacagacaacagaaccccataccagtaggcaacatttacaggagatggatccaactggggttgcaaaaatgtgtcagaatgtataacccaacaaacattctagatgtaaaacaagggccaaaagagccatttcagagctatgtagacaggttctacaaaagtttaagagcagaacagacagatgcagcagtaaagaattggatgactcaaacactgctgattcaaaatgctaacccagattgcaagc tagtgctgaaggggttggg.

In certain embodiments, the Pol protein is encoded by the nucleic acidsequence as set forth in SEQ ID NO: 8.

(5′-3′ xHIV Pol protein nt, SEQ ID NO: 8)tttttagggaagatctggccttcccacaagggaaggccagggaattttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtttggggaagagacaacaactccctctcagaagcaggagccgatagacaaggaactgtatcctttagcttccctcagatcactctttggcagcgacccctcgtcacaataaagataggggggcaattaaaggaagctctattagatacaggagcagatgatacagtattagaagaaatgaatttgccaggaagatggaaaccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgatcagatactcatagaaatctgcggacataaagctataggtacagtattagtaggacctacacctgtcaacataattggaagaaatctgttgactcagattggctgcactttaaattttcccattagtcctattgagactgtaccagtaaaattaaagccaggaatggatggcccaaaagttaaacaatggccattgacagaagaaaaaataaaagcattagtagaaatttgtacagaaatggaaaaggaaggaaaaatttcaaaaattgggcctgaaaatccatacaatactccagtatttgccataaagaaaaaagacagtactaaatggagaaaattagtagatttcagagaacttaataagagaactcaagatttctgggaagttcaattaggaataccacatcctgcagggttaaaacagaaaaaatcagtaacagtactggatgtgggcgatgcatatttttcagttcccttagataaagacttcaggaagtatactgcatttaccatacctagtataaacaatgagacaccagggattagatatcagtacaatgtgcttccacagggatggaaaggatcaccagcaatattccagtgtagcatgacaaaaatcttagagccttttagaaaacaaaatccagacatagtcatctatcaatacatggatgatttgtatgtaggatctgacttagaaatagggcagcatagaacaaaaatagaggaactgagacaacatctgttgaggtggggatttaccacaccagacaaaaaacatcagaaagaacctccattcctttggatgggttatgaactccatcctgataaatggacagtacagcctatagtgctgccagaaaaggacagctggactgtcaatgacatacagaaattagtgggaaaattgaattgggcaagtcagatttatgcagggattaaagtaaggcaattatgtaaacttcttaggggaaccaaagcactaacagaagtagtaccactaacagaagaagcagagctagaactggcagaaaacagggagattctaaaagaaccggtacatggagtgtattatgacccatcaaaagacttaatagcagaaatacagaagcaggggcaaggccaatggacatatcaaatttatcaagagccatttaaaaatctgaaaacaggaaagtatgcaagaatgaagggtgcccacactaatgatgtgaaacaattaacagaggcagtacaaaaaatagccacagaaagcatagtaatatggggaaagactcctaaatttaaattacccatacaaaaggaaacatgggaagcatggtggacagagtattggcaagccacctggattcctgagtgggagtttgtcaatacccctcccttagtgaagttatggtaccagttagagaaagaacccataataggagcagaaactttctatgtagatggggcagccaatagggaaactaaattaggaaaagcaggatatgtaactgacagaggaagacaaaaagttgtccccctaacggacacaacaaatcagaagactgagttacaagcaattcatctagctttgcaggattcgggattagaagtaaacatagtgacagactcacaatatgcattgggaatcattcaagcacaaccagataagagtgaatcagagttagtcagtcaaataatagagcagttaataaaaaaggaaaaagtctacctggcatgggtaccagcacacaaaggaattggaggaaatgaacaagtagataaattggtcagtgctggaatcaggaaagtactatttttagatggaatagataaggcccaagaagaacatgagaaatatcacagtaattggagagcaatggctagtgattttaacctaccacctgtagtagcaaaagaaatagtagccagctgtgataaatgtcagctaaaaggggaagccatgcatggacaagtagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggaaaggaccagcaaagctcctctggaaaggtgaaggggcagtagtaatacaagataatagtgacataaaagtagtgccaagaagaaaagcaaagatcatcagggattatggaaaacagatggcaggtgatgattgtgtggcaagtagacaggatgaggattaa.

In certain embodiments, the nucleotide sequence encoding gag proteinencodes a protein comprising a sequence as set forth in SEQ ID NO: 9.

(SEQ ID NO: 9) MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKIEEEQNKSKKKAQQAAADTGNNSQVSQNYPVQQIGGNYVHLPLSPRTLNAWVKLIEEKKFGAEVVPGFQALSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEVADWDLQHPQPAPQQGQLREPSGSDIAGTTSSVDEQIQWMYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNILDVKQGPKEPFQSYVDRFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGPGATLEEMNITACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ.

In certain embodiments, the nucleotide sequence encoding Pol proteinencodes a protein comprising a sequence as set forth as set forth in SEQID NO: 10.

(SEQ ID NO: 10) MNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVGDAYFSVPLDKDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMA GDDCVASRQDED.

By “complete protein or polypeptide” is meant to refer a whole orfunctional protein or polypeptide. For example, two separate portions ofa protein, which would not be functional on their own, can bind togetheror interact with one another to form a complete protein that isfunctional. The term should be construed broadly to include any type ofprotein or polypeptide known in the art. A “complete” protein does nothave to have a complete sequence of a particular domain. For example, asprovided herein the protein that can be formed by the first and secondportion can be a chimeric antigen receptor. In some embodiments, theextracellular domain of the CAR can be an antigen binding domain thatbinds to an antigen on a target cell. The antigen binding domain can be,for example, a heavy and light chain antibody molecule or can be a scFvdomain, but these can differ from a native sequence that would functionindependently of a CAR.

In certain embodiments, the polypeptide of interest is a chimericantigen receptor (CAR). The polypeptide of interest can comprise any ofthe CARs disclosed herein. In certain embodiments, the CAR comprises anextracellular (antigen binding) domain, a transmembrane domain, and anintracellular signaling domain.

In certain embodiments, the extracellular/antigen binding domain is adomain that binds a tumor antigen (e.g. anti-CD19 scFv, anti-CD19antibody, anti-CD33 scFv, and the like). In some embodiments,polypeptide of interest comprises the transmembrane domain and the CD3zeta domain. In some embodiments, the CAR comprises an intracellular4-1BB domain.

In some embodiments, extracellular/antigen binding domain is a domainthat binds to CD20, CD22, CD123, CD38, CD19, BCMA, CD33, or CD79b.

In certain embodiments, the first portion of the protein comprises anextracellular/antigen binding domain that binds a tumor antigen (e.g.anti-CD19 scFv, anti-CD19 antibody, anti-CD33 scFv, and the like), andthe second portion of the protein comprises a transmembrane domain and aCD3 zeta domain. In certain embodiments, the first portion of theprotein comprises an extracellular domain that binds a tumor antigen,and the second portion of the protein comprises a transmembrane domain,a CD3 zeta domain, and a 4-1BB domain. In certain embodiments, the firstportion of the protein comprises an extracellular domain that binds atumor antigen and a transmembrane domain, and the second portion of theprotein comprises a CD3 zeta domain. In certain embodiments, the firstportion of the protein comprises an extracellular domain that binds atumor antigen and a transmembrane domain, and the second portion of theprotein comprises a CD3 zeta domain and a 4-1BB domain. In certainembodiments, the first portion of the protein comprises an extracellulardomain that binds a tumor antigen, a transmembrane domain, and a CD3zeta domain, and the second portion of the protein comprises and a 4-1BBdomain. In certain embodiments, the first portion of the proteincomprises an extracellular domain that binds a tumor antigen, atransmembrane domain, and a 4-1BB domain, and the second portion of theprotein comprises and a CD3 zeta domain.

In certain embodiments, the polypeptide of interest is a hemoglobin betachain. These examples of proteins or polypeptides of interest arenon-limiting and any polypeptide of interest can be encoded for by thecompositions provided for herein.

Viruses

In certain embodiments, the virus particle is an adenovirus. Adenovirusparticles are based on adenoviruses, which have a low capacity forintegration into genomic DNA but a high efficiency for transfecting hostcells. Adenovirus particles contain adenovirus sequences sufficient to:(a) support packaging of the expression vector and (b) to ultimatelyexpress the protein in the host cell. In some embodiments, theadenovirus genome is a 36 kb, linear, double stranded DNA, where aforeign DNA sequence (e.g., a nucleic acid encoding a CAR) may beinserted to substitute large pieces of adenoviral DNA in order to makethe expression vector (see, e.g., Danthinne and Imperiale, Gene Therapy(2000) 7(20): 1707-1714).

In certain embodiments, the virus particle is a dependovirus, such as aparvovirus. A non-limiting example of a dependovirus is the adenoassociated virus (AAV), which takes advantage of the adenovirus coupledsystems. This AAV expression vector has a high frequency of integrationinto the host genome. It can infect nondividing cells, thus making ituseful for delivery of genes into mammalian cells, for example, intissue cultures or in vivo. The AAV has a broad host range forinfectivity. Details concerning the generation and use of AAV aredescribed in U.S. Pat. Nos. 5,139,941 and 4,797,368, each of which isincorporated by reference in its entirety. In some embodiments, the AAVparticle is an AAV9 particle. An example of a AAV9 particle is providedin U.S. Pat. No. 7,906,111, which is hereby incorporated by reference inits entirety.

Retrovirus expression vectors are capable of integrating into the hostgenome, delivering a large amount of foreign genetic material, infectinga broad spectrum of species and cell types and being packaged in specialcell lines. The retroviral particle is constructed by inserting anucleic acid (e.g., a nucleic acid encoding a CAR) into the viral genomeat certain locations to produce a virus that is replication defective.Though the retroviral particles are able to infect a broad variety ofcell types, integration and stable expression of the CAR requires thedivision of host cells.

In certain embodiments, the virus particle is a lentiviral particle.Lentiviral particles are derived from lentiviruses, which areretroviruses that, in addition to the common retroviral genes gag, pol,and env, contain other genes with regulatory or structural function(see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2)and the Simian Immunodeficiency Virus (SIV). Lentiviral particles havebeen generated by multiply attenuating the HIV virulence genes, forexample, the genes env, vif, vpr, vpu and nef are deleted making thevector biologically safe. Lentiviral particles are capable of infectingnon-dividing cells and can be used for both in vivo and ex vivo genetransfer and expression, e.g., of a nucleic acid encoding a CAR (see,e.g., U.S. Pat. No. 5,994,136).

Pseudotyped Viral Particles/Targeting Moiety

To confer specificity of the viral particles to target cells, viralparticles can be pseudotyped. Capsid proteins and envelope glycoproteinsare implicated in virus attachment and interactions with cellularreceptors, determining cell tropism. Manipulation of these viral surfaceproteins therefore may improve the transduction capacity of thesevectors, expanding or restricting their tropism. Furthermore,experiments with vector pseudotyping demonstrated that pseudotypedvectors could achieve higher transduction titers and increasetransduction efficacy.

In some embodiments, a virus particle comprising a targeting moiety thatbinds to a target on a cell and a nucleic acid molecule that encodes apolypeptide of interest is provided.

In some embodiments, a virus particle comprising a first targetingmoiety that binds to a first target on a cell and a nucleic acidmolecule that encodes a first portion of a protein or polypeptide areprovided. In some embodiments, a virus particle comprising a secondtargeting moiety that binds to a second target on the cell and a nucleicacid molecule that encodes for a second portion of the protein or thepolypeptide are provided.

In some embodiments, a composition comprising a first viral particlehaving a first targeting moiety that binds to a first target on a cell,and a second viral particle having a second targeting moiety that bindsto a second target on the cell are provided. In certain embodiments, thefirst target and the second target are different.

The targeting moiety can be any type of targeting moeity, including butnot limited to, an antigen binding domain, a DARPIN, a FN3 domain, anantibody, a Centryn, Stem Cell Factor protein (SCF, KIT-ligand, KL, orsteel factor). In certain embodiments, the target is cKit (CD117), CD4,CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta,CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, and CXCR3. In certain embodiments, the target iscKit (CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta,TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, or CD68.

In certain embodiments, the first target and the second target are eachindependently selected from the group consisting of cKit (CD117), CD4,CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta,CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, and CXCR3. In certain embodiments, the firsttarget is cKit (CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha,TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, or CD68.In certain embodiments, the second target is CCR7, CD62L, CD25, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3.

In certain embodiments, the targeting moiety (first and/or second) is aprotein that binds to a target, an antigen binding domain, an antibody,a scFv, a DARPIN, and a FN3 domain, or any combination thereof.

In certain embodiments, the targeting moiety (first and/or second) isStem Cell Factor protein (SCF, KIT-ligand, KL, or steel factor) or amoiety that binds to cKit (CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2,TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33,CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, orCXCR3. In some embodiments, the targeting moiety binds to CD4 and/orCD8. In some embodiments, the targeting moiety binds to CD20, CD22,CD123, CD38, CD19, BCMA, CD33, or CD79b.

In certain embodiments, the engineered virus particle is a pseudotypedvirus particle. In certain embodiments, the engineered virus particle isa pseudotyped lentiviral virus particle, an adenovirus, or anadeno-associated virus. In certain embodiments, the pseudotypedlentiviral virus particle is pseudotyped with a morbillivirus, such as ameasles virus, glycoprotein and/or a Nipah virus glycoprotein. Incertain embodiments, the engineered virus particle is pseudotyped with aF protein or H protein of a morbillivirus. In some embodiments, themeasles F protein or H protein is mutated. The mutations can be used insubjects that have been exposed to the measles virus or measles vaccine,which can avoid neutralization by anti-MV antibodies that may be presentin those that have been exposed to the measles virus or the measlesvaccine. In some embodiments, the engineered virus particle ispseudotyped with a F protein or G protein of a Nipah virus.

Examples of the F, G, or H proteins can be found in U.S. Pat. No.10,415,057, which is hereby incorporated by reference in its entirety.In some embodiments, the pseudotyped viral particle comprises a fusion(F) and a hemagglutinin (H) protein of a morbillivirus, wherein thecytoplasmic portions of said F and H proteins are truncated and whereinthe truncated cytoplasmic portion of the F protein comprises at least 1positively charged amino acid residue and the truncated cytoplasmicportion of the H protein is truncated to allow efficient pseudotypingand has fusion support function. In some embodiments, the morbillivirusis a measles virus, or the Edmonston strain of measles virus. In someembodiments, the truncated cytoplasmic portion of the H proteincomprises at least 9 consecutive amino acid residues of the C-terminalcytoplasmic portion of the H protein plus an additional methinonine atthe N-terminus. In some embodiments, the truncated cytoplasmic portionof the F protein comprises at least 3 consecutive amino acid residues ofthe N-terminal cytoplasmic portion of the F protein and the truncatedcytoplasmic portion of the H protein comprises at least 13 consecutiveamino acid residues of the C-terminal cytoplasmic portion of the Hprotein plus an additional methinonine at the N-terminus, wherein one tofour of the N-terminal amino acid residues of said at least 13consecutive amino acid residues of the C-terminal cytoplasmic portion ofthe H protein can be replaced by alanine residues. In some embodiments,the truncated F protein is FcΔ24 or FcΔ30 and/or the truncated H proteinis selected from the group consisting of HcΔ14, HcΔ15, HcΔ16, HcΔ17,HcΔ18, Hc.DELTA.19, HcΔ20, HcΔ21+A and HcΔ24+4A. These proteins are alsodescribed in U.S. Pat. No. 10,415,057, which is hereby incorporated byreference in its entirety.

In some embodiments, the viral particle comprises a measles virus Fprotein, which can be referred to as Δ30, having an amino acid sequenceof:

(SEQ ID NO: 11) MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTCIVYILI AVCLGGLIGIPALICCCRGR.

In some embodiments, the viral particle comprises a measles virus Hprotein, which can be referred to as Δ18+4A, having an amino acidsequence of:

(SEQ ID NO: 12) MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPALFNVPIKEAGEDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSAVEHAVVYYVYSPSRLSSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNRRG G.Without being bound to any theory, the 4 amino acid changes are used toinactivate the natural tropism for CD46.

In some embodiments, the viral particle comprises the measles Δ30 (SEQID NO: 11) and the measles Δ18+4A (SEQ ID NO: 12) amino acid sequences.

In some embodiments, the viral particle comprises a Nipah virus Fprotein, which can be referred to as Δ30, having the amino acid sequenceof:

(SEQ ID NO: 13) MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNT.

In some embodiments, the viral particle comprises a Nipah virus Gprotein, which can be referred to as Δ34+4A, having the amino acidsequence of:

(SEQ ID NO: 14) MKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIMIIQNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKPKSNGGGYNQHQLALRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVLTVNPLVVNWRNNTVISRPGQSQCPRFNTCPAICAEGVYNDAFLIDRINWISAGVFLDSNATAANPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDT GDNVIRPKLFAVKIPEQCT.

Without being bound to any theory, the 4 amino acid changes are used toinactivate the natural tropism for Ephrin.

In some embodiments, In some embodiments, the viral particle comprisesthe Nipah Δ30 (SEQ ID NO: 13) and the Nipah Δ18+4A (SEQ ID NO: 14) aminoacid sequences.

In some embodiments, the H protein is a fusion of HmutΔ18, HmutΔ19 orHmutΔ24+4A and a single chain antibody or a ligand to a cell surfacemarker at its ectodomain. In some embodiments, the single chain antibodyis directed against the cell surface proteins as provided herein,including but not limited to, CD20 (scFvCD20), CD34 (scFvCD34), VEGFR-2(scFvA7), CD133 (scFvCD133), or the ligand is EGF (the ligand of theEGF-receptor). In some embodiments, the H protein is a fusion ofHmutΔ18, HmutΔ19 or HmutΔ24+4A fused to scFvCD20, scFvCD34, scFvA7, EGFor scFvCD133 and the F protein is FcΔ30 or FcΔ24. In some embodimentsthe truncated H protein is a fusion defined by the amino acid sequenceset forth in SEQ ID NO: 2 or SEQ ID NO: 4 of U.S. Pat. No. 10,415,057,which is hereby incorporated by reference in its entirety i.e., SEQ IDNOs: 15 and 16 of the present disclosure, respectively:

(SEQ ID NO: 15) MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPALFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSAVEHAVVYYVYSPSRLSSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNAAQPAIEGRMAQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSKISGGGGSGGGGSGGGGSGGSSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAG TKLELKAAARGSHHHHHH.(SEQ ID NO: 16) MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPALFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSAVEHAVVYYVYSPSRLSSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNAAQPAMANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRD LKWWELRAAARGSHHHHHH.

In some embodiments, the first virus particle and the second virusparticle are pseudotyped with the same envelope protein. In someembodiments, the first viral vector and the second virus particle arepseudotyped with different envelope proteins. In some embodiments, thecompositions provided herein comprise an engingeered virus particlecomprising targeting moieties that bind a cell. In some embodiments, thecompositions provided herein comprise a first and second virus particlecomprising targeting moieties that bind a first and second target on acell. The cell can be any type of cell including, but not limited to, aT cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T cell, agamma-delta T cell, a lymphoid progenitor cell, a hematopoietic stemcell, a myeloid cell, a monocyte, a macrophage, a central memory T cell,a naïve T cell, an activated T cell, a regulatory T Cell (Treg), or aT-Cell^(CD8+CCR7+). In some embodiments, the cell is a CD4+CD8+ T cell.

Accordingly, in some embodiments an engineered viral particle isprovided comprising an engineered envelope comprising a polypeptidehaving the amino acid sequence of SEQ ID NO: 9. In some embodiments, theengineered viral particle comprises a heterologous polypeptide targetingmoiety for binding to a target cell. In some embodiments, the viralparticle comprises a nucleic acid molecule encoding a heterologouspolypeptide of interest. In some embodiments, the targeting moiety isfused to a mutated fusion protein present on the surface of theengineered viral particle. In some embodiments, the viral particle is alentivirus pseudotyped with a) a measles virus (MV) hemagglutinin (HA)protein and/or an MV fusion (F) protein (MV-F protein) and wherein theMV-HA protein or the MV-F protein comprises a mutation or a mutatedbinding domain compared to its naturally occurring protein; or b) aNipah virus F protein and/or a Nipah virus G protein and wherein theNipah virus F protein and/or a Nipah virus G protein comprises amutation or a mutated binding domain compared to its naturally occurringprotein. In some embodiments, the targeting moiety is fused to the MV-HAprotein and/or the MV-F protein. In some embodiments, the targetingmoiety is fused to the Nipah virus F protein and/or a Nipah virus Gprotein. In some embodiments, the targeting moiety is a scFv, an antigenbinding domain, a DARPIN, a VHH, or a FN3 domain. In some embodiments,the targeting moiety binds to protein selected from the group consistingof Stem Cell Factor protein (SCF, KIT-ligand, KL, or steel factor) or amoiety that binds to cKit (CDl 17), CD4, CD8, CD3, CD3D, CD3E, CD3G,CD3Z, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta,CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, and CXCR3. In some embodiments, the heterologouspolypeptide of interest is a chimeric antigen receptor (CAR). In someembodiments, the chimeric antigen receptor comprises an extracellulardomain, transmembrane domain, and an intracellular signaling domain. Insome embodiments, the extracellular domain binds to CD20, CD22, CD123,CD38, CD19, BCMA, CD33, or CD79b. In some embodiments, the heterologouspolypeptide of interest is a hemoglobin beta chain.

Cells

In some embodiments, the embodiments provided include a cell comprisingthe engineered virus particle or particles as provided herein. In someembodiments, the cell comprises the polypeptide of interest encoded bythe engineered virus particle.

In certain embodiments, the polypeptide of interest is a hemoglobin betachain. In certain embodiments, the polypeptide of interest is aheterologous chimeric antigen receptor (CAR).

In certain embodiments, the cell is a T cell, e.g., a CD8+ T cell (e.g.,a CD8+ naive T cell, central memory T cell, or effector memory T cell),a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell(Treg), an alpha-beta T cell, a gamma-delta T cell, a stem cell memory Tcell, a central memory T cell, a naïve T cell, an activated T cell, aT-Cell^(CD8+CCR7+), a lymphoid progenitor cell, a hematopoietic stemcell, a natural killer cell (NK cell), or a dendritic cell. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils. In an embodiment, the target cell is aninduced pluripotent stem (iPS) cell or a cell derived from an iPS cell,e.g., an iPS cell generated from a subject, manipulated to alter (e.g.,induce a mutation in) or manipulate the expression of one or more targetgenes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell(e.g., a CD8+ naive T cell, central memory T cell, or effector memory Tcell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitorcell or a hematopoietic stem cell. In some embodiments, the cell is aCD4+CD8+ T cell.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. Among the sub-types and subpopulations of Tcells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells,effector T cells (TEFF), memory T cells and sub-types thereof, such asstem cell memory T (TSCM), central memory T (TCM), effector memory T(TEM), or terminally differentiated effector memory T cells,tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells,helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT)cells, naturally occurring and adaptive regulatory T (Treg) cells,helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9cells, TH22 cells, follicular helper T cells, alpha/beta T cells, anddelta/gamma T cells. In certain embodiments, any number of T cell linesavailable in the art, may be used.

Viral Vector

Provided herein are compositions that can be used, for example, toselectively transduce a cell expressing specific cell surface markers invivo using a viral vector. In some embodiments, the vector encodes thepolypeptide of interest. In some embodiments, the viral vector is apseudotyped vector.

In some embodiments, the pseudotyped viral vector comprises a chimericgag protein, as described herein. In some embodiments, the chimeric gagprotein is referred to as xHIV gag protein. In some embodiments, the gagprotein comprises the sequence as set forth in SEQ ID NO: 9. In someembodiments, the viral vector comprises a polynucleotide moleculeencoding the chimeric gag protein. In some embodiments, thepolynucleotide molecule encoding the chimeric gag protein comprises asequence of SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, thenucleotide sequence further comprises a long-terminal repeat (LTR), aSIV element, a sequence encoding a Pol protein, and/or a sequenceencoding an Envelope (Env) polyprotein. In some embodiments, the gagprotein, the SIV element, and the Pol protein of the xHIV are encoded bythe nucleic acid sequence as set forth in SEQ ID NO: 5. In someembodiments, the gag protein is encoded by the nucleic acid sequence asset forth in SEQ ID NO: 6. In some embodiments, the SIV element isencoded by the nucleic acid sequence as set forth in SEQ ID NO: 7. Insome embodiments, the nucleic acid sequence encoding the SIV element islocated within the nucleic acid sequence encoding the gag protein as setforth in SEQ ID NO: 6. In some embodiments, the Pol protein is encodedby the nucleic acid sequence as set forth in SEQ ID NO: 8. In someembodiments, the polynucleic acid molecule encoding the Pol proteinencodes for a protein comprising the sequence as set forth in SEQ ID NO:10.

In some embodiments, a viral particle is provided comprising a chimericgag protein, as described herein. In some embodiments, the chimeric gagprotein comprises an amino acid sequence as set forth in SEQ ID NO: 9.In some embodiments, a viral particle comprising the chimeric gagprotein comprises a heterologous transgene of interest, such as achimeric antigen receptor. Non-limiting examples of chimeric antigenreceptors are provided herein.

Also provided herein are compositions that can be used, for example, toselectively transduce a cell expressing specific cell surface markers invivo using a plurality of viral vectors, which can be referred to as asplit viral vector system. In some embodiments, one vector encodes afirst portion of the molecule of interest, and another vector encodes asecond portion of the molecule of interest. In certain embodiments, afirst portion and a second portion of a protein (or nucleic acidsencoding said proteins) are expressed by, or encoded by a plurality ofvectors. In some embodiments, the first portion and the second portionare expressed by, or encoded by, 2, 3, or 4 different vectors. For theavoidance of doubt, the vectors are considered different if they encodefor different portions that have different sequences or the molecule ofinterest, even if the molecule's sequence or structure are similar oroverlap.

In some embodiments, one viral particle provides a viral vector encodinga first portion of a molecule of interest, and another viral particleprovides a second viral vector encoding a second portion of a moleculeof interest. As provided herein, in some embodiments, a molecule ofinterest is a chimeric antigen receptor.

In some embodiments, a composition comprising a first viral particle anda second viral particle are provided. In some embodiments, the firstviral particle comprises a first targeting moiety that binds to a firsttarget on a cell and a nucleic acid molecule that encodes a firstportion of a protein or polypeptide. In some embodiments, the secondviral particle comprises a second targeting moiety that binds to asecond target on the cell and a nucleic acid molecule that encodes asecond portion of the protein or the polypeptide. Once expressed in thecell, the first and second portions of the protein or the polypeptidecan bind together or interact with one another to form a complete orfunctional protein or polypeptide.

Expression vectors comprising a nucleic acid of the present disclosurecan be introduced into a host cell by any method or composition known topersons skilled in the art. The expression vectors may include viralsequences for transfection, if desired. Alternatively, the expressionvectors may be introduced by fusion, electroporation, biolistics,transfection, lipofection, or the like. Although the cell can betransduced or transfected in vivo, in some embodiments, the transducedcells can then be isolated from the subject and then, in someembodiments, may be grown and expanded in culture ex vivo. The expandedcells can then be screened by virtue of a marker present in the vectors.The expanded cells can then be reintroduced into the same subject or adifferent subject for treatment. Various markers that may be used areknown in the art, and may include hprt, neomycin resistance, thymidinekinase, hygromycin resistance, etc. In some embodiments, the host cellis an immune cell or precursor thereof, e.g., a T cell, an NK cell, oran NKT cell.

Embodiments provided herein also include nucleic acids encoding any ofthe virus particles, in any of the embodiments, disclosed herein.

In some embodiments, a nucleic acid of the present disclosure isprovided for the production of a protein (e.g. CAR) as described herein,e.g., in a cell or in a subject in vivo.

In some embodiments, a nucleic acid of the present disclosure may beoperably linked to a transcriptional control element, e.g., a promoter,and enhancer, etc. Suitable promoter and enhancer elements are known tothose of skill in the art. In certain embodiments, the nucleic acidencoding a CAR is in operable linkage with a promoter. In certainembodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

For expression in a eukaryotic cell, suitable promoters include, but arenot limited to, light and/or heavy chain immunoglobulin gene promoterand enhancer elements; cytomegalovirus immediate early promoter; herpessimplex virus thymidine kinase promoter; early and late SV40 promoters;promoter present in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters. Suitable reversible promoters, including reversible induciblepromoters are known in the art. Such reversible promoters may beisolated and derived from many organisms, e.g., eukaryotes andprokaryotes. Modification of reversible promoters derived from a firstorganism for use in a second organism, e.g., a first prokaryote and asecond a eukaryote, a first eukaryote and a second a prokaryote, etc.,is well known in the art. Such reversible promoters, and systems basedon such reversible promoters but also comprising additional controlproteins, include, but are not limited to, alcohol regulated promoters(e.g., alcohol dehydrogenase I (alcA) gene promoter, promotersresponsive to alcohol transactivator proteins (A1cR), etc.),tetracycline regulated promoters, (e.g., promoter systems includingTetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g.,rat glucocorticoid receptor promoter systems, human estrogen receptorpromoter systems, retinoid promoter systems, thyroid promoter systems,ecdysone promoter systems, mifepristone promoter systems, etc.), metalregulated promoters (e.g., metallothionein promoter systems, etc.),pathogenesis-related regulated promoters (e.g., salicylic acid regulatedpromoters, ethylene regulated promoters, benzothiadiazole regulatedpromoters, etc.), temperature regulated promoters (e.g., heat shockinducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter,etc.), light regulated promoters, synthetic inducible promoters, and thelike.

In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an NcrI (p46) promoter; see, e.g., Eckelhart et al. Blood (2011)117:1565.

Other examples of suitable promoters include the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to a simian virus 40 (SV40) early promoter, a mouse mammarytumor virus (MMTV) or human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, a MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the EF-1 alpha promoter, as well as human gene promoterssuch as, but not limited to, an actin promoter, a myosin promoter, ahemoglobin promoter, and a creatine kinase promoter. Further, thevectors should not be limited to the use of constitutive promoters.Inducible promoters can also be used. The use of an inducible promoterprovides a molecular switch capable of turning on expression of thepolynucleotide sequence which it is operatively linked when suchexpression is desired, or turning off the expression when expression isnot desired. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter.

In some embodiments, the locus or construct or transgene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99,the disclosure of which is incorporated herein by reference). Anysuitable combination of recombinase, endonuclease, ligase, recombinationsites, etc. known to the art may be used in generating an irreversiblyswitchable promoter. Methods, mechanisms, and requirements forperforming site-specific recombination, described elsewhere herein, finduse in generating irreversibly switched promoters and are well known inthe art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006)567-605; and Tropp, Molecular Biology (2012) (Jones & BartlettPublishers, Sudbury, Mass.), the disclosures of which are incorporatedherein by reference.

A nucleic acid of the present disclosure may be present within anexpression vector and/or a cloning vector. An expression vector caninclude a selectable marker, an origin of replication, and otherfeatures that provide for replication and/or maintenance of the vector.Suitable expression vectors include, e.g., plasmids, viral vectors, andthe like. Large numbers of suitable vectors and promoters are known tothose of skill in the art; many are commercially available forgenerating a subject recombinant construct. The following vectors areprovided by way of example, and should not be construed in anyway aslimiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest.Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther.(1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995)92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. GeneTher. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA(1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci.(1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690,Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum.Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus(see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); aretroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus,and vectors derived from retroviruses such as Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, human immunodeficiency virus,myeloproliferative sarcoma virus, and mammary tumor virus); and thelike.

Additional expression vectors suitable for use are, e.g., withoutlimitation, a lentivirus vector, a gamma retrovirus vector, a foamyvirus vector, an adeno-associated virus vector, an adenovirus vector, apox virus vector, a herpes virus vector, an engineered hybrid virusvector, a transposon mediated vector, and the like. Viruses, which areuseful as vectors include, but are not limited to, retroviruses,adenoviruses, adeno-associated viruses, herpes viruses, andlentiviruses.

In some embodiments, an expression vector (e.g., a lentiviral vector)may be used to introduce the polypeptide of interest (e.g., CAR, or aportion thereof) into a cell (e.g., a T cell). Accordingly, anexpression vector (e.g., a lentiviral vector) may comprise a nucleicacid encoding for a polypeptide of interest (e.g., CAR, or a portionthereof). As provided herein, the polypeptide of interest can beintroduced into the cell through the use of expression vectors. In someembodiments, the expression vector (e.g., lentiviral vector) willcomprise additional elements that will aid in the functional expressionof the protein or polypeptide (e.g., CAR, or a portion thereof) encodedtherein. In some embodiments, an expression vector comprising a nucleicacid encoding a polypeptide of interest (e.g., CAR, or a portionthereof) further comprises a mammalian promoter. In some embodiments,the vector further comprises an elongation-factor-1-alpha promoter(EF-1α promoter). Use of an EF-1α promoter may increase the efficiencyin expression of downstream transgenes (e.g., a protein or polypeptide(e.g., CAR, or a portion thereof) encoding nucleic acid sequence).Physiologic promoters (e.g., an EF-1α promoter) may be less likely toinduce integration mediated genotoxicity, and may abrogate the abilityof the retroviral vector to transform stem cells. Other physiologicalpromoters suitable for use in a vector (e.g., lentiviral vector) areknown to those of skill in the art and may be incorporated into a vectorof the present invention. In some embodiments, the vector (e.g.,lentiviral vector) further comprises a non-requisite cis acting sequencethat may improve titers and gene expression. One non-limiting example ofa non-requisite cis acting sequence is the central polypurine tract andcentral termination sequence (cPPT/CTS) which is important for efficientreverse transcription and nuclear import. Other non-requisite cis actingsequences are known to those of skill in the art and may be incorporatedinto a vector (e.g., lentiviral vector) of the present invention. Insome embodiments, the vector further comprises a posttranscriptionalregulatory element. Posttranscriptional regulatory elements may improveRNA translation, improve transgene expression and stabilize RNAtranscripts. One example of a posttranscriptional regulatory element isthe woodchuck hepatitis virus posttranscriptional regulatory element(WPRE). Accordingly, in some embodiments a vector for the presentinvention further comprises a WPRE sequence. Various posttranscriptionalregulator elements are known to those of skill in the art and may beincorporated into a vector (e.g., lentiviral vector) of the presentinvention. A vector of the present invention may further compriseadditional elements such as a rev response element (RRE) for RNAtransport, packaging sequences, and 5′ and 3′ long terminal repeats(LTRs). The term “long terminal repeat” or “LTR” refers to domains ofbase pairs located at the ends of retroviral DNAs which comprise U3, Rand U5 regions. LTRs generally provide functions required for theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. In someembodiments, a vector (e.g., lentiviral vector) of the present inventionincludes a 3′ U3 deleted LTR. Accordingly, a vector (e.g., lentiviralvector) of the present invention may comprise any combination of theelements described herein to enhance the efficiency of functionalexpression of transgenes. For example, a vector (e.g., lentiviralvector) of the present invention may comprise a WPRE sequence, cPPTsequence, RRE sequence, in addition to a nucleic acid encoding for aprotein or polypeptide (e.g., CAR).

In some embodiments, the vector is a self-inactivating vector. As usedherein, the term “self-inactivating vector” refers to vectors in whichthe 3′ LTR enhancer promoter region (U3 region) has been modified (e.g.,by deletion or substitution). A self-inactivating vector may preventviral transcription beyond the first round of viral replication.Consequently, a self-inactivating vector may be capable of infecting andthen integrating into a host genome (e.g., a mammalian genome) onlyonce, and cannot be passed further. Accordingly, self-inactivatingvectors may greatly reduce the risk of creating a replication-competentvirus.

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell may also containeither a selectable marker gene or a reporter gene, or both, tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In some embodiments, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection or co-transductionprocedure. Both selectable markers and reporter genes may be flankedwith appropriate regulatory sequences to enable expression in the hostcells. Useful selectable markers include, without limitation,antibiotic-resistance genes. Reporter genes are used for identifyingpotentially transfected cells and for evaluating the functionality ofregulatory sequences. In general, a reporter gene is a gene that is notpresent in or expressed by the recipient organism or tissue and thatencodes a polypeptide whose expression is manifested by some easilydetectable property, e.g., enzymatic activity. Expression of thereporter gene is assessed at a suitable time after the DNA has beenintroduced into the recipient cells. Suitable reporter genes mayinclude, without limitation, genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

Dimerizing Agent

In certain embodiments, the first and second portion of the protein orpolypeptide form a complete protein in the presence of a dimerizingagent. Any dimerizing agent known to one of ordinary skill in the artcan be used, including but not limited to, rimiducid((1R)-3-(3,4-dimethoxyphenyl)-1-[3-({[2-(2-{3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyloxy]propyl]phenoxy}acetamido)ethyl]carbamoyl}methoxy)phenyl]propyl(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate(AP1903)), erythropoietin (EPO), thrombopoietin, or rapamycin, oranalogues thereof. For example, the protein of interest that is splitinto separate portions that that can then be dimerized will contain aerythropoietin binding domain, thrombopoietin binding domain, orrapamycin, or analogoues thereof, binding domain, such that when thecompounds are given the compound will facilitate the joining of theseparate portions of the protein to form the complete protein. In someembodiments, a compound used to induce erythropoietin or thrombopoietinis administered, as opposed to erythropoietin or thrombopoietinthemselves, to induce the production of erythropoietin or thrombopoietinin vivo, which will join the portions of the protein to form thecomplete protein.

In certain embodiments, the first and second portions of the protein orpolypeptide can bind together through the excision of an intein domain.An intein is a protein domain that can spontaneously splice its flankingN- and C-terminal domains to become a mature protein and excise itselffrom a sequence. Similar to introns at the mRNA level, the protein is anintron-like protein and is therefore named intein. Embodiments ofinteins can be found in the literature, see, for example, U.S. Pat. Nos.10,407,742, and 10,087,213, which are incorporated by reference in theirentirety herein.

Chimeric Antigen Receptors (CARs)

The present invention provides, among other things, compositions andmethods for transducing cells (e.g. T cells) in vivo. In someembodiments, these compositions and methods are performed or usedwithout culturing or transducing the cells ex-vivo. In some embodiments,a patient with a malignancy requiring CAR T cell therapy can be treatedwith an off-the-shelf composition that transduces their T cells in vivo,which generates CAR T cells in situ. The compositions and methods of thepresent invention, among other things, (i) are able to specificallytransduce the target cells of interest, (ii) have high transductionefficiency to lead to a sufficient number of T cells carrying thetransgene after in vivo administration, and (iii) result in transducedcells that are functional.

Certain embodiments of the invention include compositions comprising apolypeptide of interest that is a chimeric antigen receptor (CAR). CARsof the present invention comprise an extracellular (antigen binding)domain, a transmembrane domain, and an intracellular signaling domain.The intracellular signaling domain comprises a stimulatory domain, andoptionally, a co-stimulatory domain.

The antigen binding domain may be operably linked to another domain ofthe CAR, such as the transmembrane domain or the intracellular domain,both described elsewhere herein, for expression in the cell. In oneembodiment, a first nucleic acid sequence encoding the antigen bindingdomain is operably linked to a second nucleic acid encoding atransmembrane domain, and further operably linked to a third a nucleicacid sequence encoding an intracellular signaling domain.

The antigen binding domains described herein can be combined with any ofthe transmembrane domains described herein, any of the intracellularsignaling domains or cytoplasmic domains described herein, or any of theother domains described herein that may be included in a CAR expressedby the vectors. In some embodiments, the CAR comprises a hinge domain,such as, but not limited to, those described herein. In someembodiments, the CAR comprises a spacer domain, such as, but not limitedto, those described herein. In some embodiments, one or more of theantigen binding domain, transmembrane domain, and intracellularsignaling domain is separated by a linker.

Extracellular/Antigen Binding Domain

The antigen binding domain of a CAR is an extracellular region of theCAR for binding to a specific target antigen including proteins,carbohydrates, and glycolipids. In some embodiments, the CAR comprisesaffinity to a target antigen on a target cell. The target antigen mayinclude any type of protein, or epitope thereof, associated with thetarget cell. For example, the CAR may comprise affinity to a targetantigen on a target cell that indicates a particular disease state ofthe target cell.

In one embodiment, the target cell antigen is a tumor associated antigen(TAA). Examples of tumor associated antigens (TAAs), include but are notlimited to, differentiation antigens such as MART-1/MelanA (MART-I),gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specificmultilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15;overexpressed embryonic antigens such as CEA; overexpressed oncogenesand mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; uniquetumor antigens resulting from chromosomal translocations; such asBCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such asthe Epstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS. In some embodiments, the antigenbinding domain of the CAR targets an antigen that includes but is notlimited to CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA,PSCA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, CD19,BCMA, CD33, or CD79b, and the like.

In some embodiments, the extracellular domain binds to CD20, CD22,CD123, CD38, CD19, BCMA, CD33, CD8, CCR7 or CD79b.

Depending on the desired antigen to be targeted, the CAR can beengineered to include the appropriate antigen binding domain that isspecific to the desired antigen target. For example, if CD19 is thedesired antigen that is to be targeted, an antibody for CD19 can be usedas the antigen bind moiety for incorporation into the CAR.

In some embodiments, the target cell antigen is CD19. As such, in someembodiments, a CAR has affinity for CD19 on a target cell. This shouldnot be construed as limiting in any way, as a CAR having affinity forany target antigen is suitable for use in any of the compositions ormethods provided for herein.

As described herein, a CAR of the present disclosure having affinity fora specific target antigen on a target cell may comprise atarget-specific binding domain. In some embodiments, the target-specificbinding domain is a murine target-specific binding domain, e.g., thetarget-specific binding domain is of murine origin. In some embodiments,the target-specific binding domain is a human target-specific bindingdomain, e.g., the target-specific binding domain is of human origin.

In some embodiments, a CAR of the present disclosure may have affinityfor one or more target antigens on one or more target cells. In someembodiments, a CAR may have affinity for one or more target antigens ona target cell. In such embodiments, the CAR is a bispecific CAR, or amultispecific CAR. In some embodiments, the CAR comprises one or moretarget-specific binding domains that confer affinity for one or moretarget antigens. In some embodiments, the CAR comprises one or moretarget-specific binding domains that confer affinity for the same targetantigen. For example, a CAR comprising one or more target-specificbinding domains having affinity for the same target antigen could binddistinct epitopes of the target antigen. When a plurality oftarget-specific binding domains is present in a CAR, the binding domainsmay be arranged in tandem and may be separated by linker peptides. Forexample, in a CAR comprising two target-specific binding domains, thebinding domains are connected to each other covalently on a singlepolypeptide chain, through an oligo- or polypeptide linker, an Fc hingeregion, or a membrane hinge region.

In some embodiments, the antigen binding domain is as provided herein.In some embodiments, the antigen binding domain is selected from thegroup consisting of an antibody, an antigen binding fragment (Fab), anda single-chain variable fragment (scFv). In some embodiments, theantigen binding domain is a VHH. In some embodiments, the antigenbinding domain is a FN3 domain. In some embodiments, the antigen bindingdomain is a DARPIN. In some embodiments, a CD19 binding domain isselected from the group consisting of a CD19-specific antibody, aCD19-specific Fab, and a CD19-specific scFv. In one embodiment, a CD19binding domain is a CD19-specific antibody. In some embodiments, a CD19binding domain is a CD19-specific Fab. In some embodiments, a CD19binding domain is a CD19-specific scFv. CD19 is just one example and anyother target antigen can be substituted for the antibody or antibodytype molecule. In some embodiments, the target antigen is CD20, CD22,CD123, CD38, CD19, BCMA, CD33, CD8, CCR7, or CD79b.

The antigen binding domain can include any domain that binds to theantigen and may include, but is not limited to, a monoclonal antibody, apolyclonal antibody, a synthetic antibody, a human antibody, a humanizedantibody, a non-human antibody, and any fragment thereof. In someembodiments, the antigen binding domain portion comprises a mammalianantibody or a fragment thereof. The choice of antigen binding domain maydepend upon the type and number of antigens that are present on thesurface of a target cell.

As used herein, the term “single-chain variable fragment” or “scFv” is afusion protein of the variable regions of the heavy (VH) and lightchains (VL) of an immunoglobulin (e.g., mouse or human) covalentlylinked to form a VH::VL heterodimer. The heavy (VH) and light chains(VL) are either joined directly or joined by a peptide-encoding linker,which connects the N-terminus of the VH with the C-terminus of the VL,or the C-terminus of the VH with the N-terminus of the VL. In someembodiments, the antigen binding domain (e.g., CD19 binding domain)comprises an scFv having the configuration from N-terminus toC-terminus, VH-linker-VL. In some embodiments, the antigen bindingdomain comprises an scFv having the configuration from N-terminus toC-terminus, VL-linker-VH.

In some embodiments, the linker is rich in glycine for flexibility, aswell as serine or threonine for solubility. The linker can link theheavy chain variable region and the light chain variable region of theextracellular antigen-binding domain. Non-limiting examples of linkersare disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014/087010, the contents of which are hereby incorporated by referencein their entireties. Various linker sequences are known in the art,including, without limitation, glycine serine (GS) linkers such as(GS)_(n), (GSGGS)_(n) (SEQ ID NO: 1), (GGGS)_(n) (SEQ ID NO: 2),(GGGGS)_(n) (SEQ ID NO: 3), (GGSGG)_(n) (SEQ ID NO:4) where each n is,independently, an integer of at least 1 or 1-5. In some embodiments, theserine of the linker is replaced with an alanine.

Despite removal of the constant regions and the introduction of alinker, scFv proteins retain the specificity of the originalimmunoglobulin. Single chain Fv polypeptide antibodies can be expressedfrom a nucleic acid comprising VH- and VL-encoding sequences asdescribed by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883,1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; andU.S. Patent Publication Nos. 20050196754 and 20050196754. AntagonisticscFvs having inhibitory activity have been described (see, e.g., Zhao etal., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J CachexiaSarcopenia Muscle 2012 Aug. 12; Shieh et al., J Imunol 2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63;Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al.,Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have beendescribed (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7;Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit RevImmunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 20031638(3):257-66).

As used herein, “Fab” refers to a fragment of an antibody structure thatbinds to an antigen but is monovalent and does not have a Fc portion,for example, an antibody digested by the enzyme papain yields two Fabfragments and an Fc fragment (e.g., a heavy (H) chain constant region;Fc region that does not bind to an antigen).

As used herein, “F(ab′)2” refers to an antibody fragment generated bypepsin digestion of whole IgG antibodies, wherein this fragment has twoantigen binding (ab′) (bivalent) regions, wherein each (ab′) regioncomprises two separate amino acid chains, a part of a H chain and alight (L) chain linked by an S—S bond for binding an antigen and wherethe remaining H chain portions are linked together. A “F(ab′)2” fragmentcan be split into two individual Fab′ fragments.

Other examples of antibodies or antigen binding domains are provided forherein and can also be used in the CAR construct.

In some embodiments, the antigen binding domain may be derived from thesame species in which the CAR will ultimately be used. For example, foruse in humans, the antigen binding domain of the CAR may comprise ahuman antibody or a fragment thereof. In some embodiments, the antigenbinding domain may be derived from a different species in which the CARwill ultimately be used. For example, for use in humans, the antigenbinding domain of the CAR may comprise a murine antibody or a fragmentthereof

Transmembrane Domain

CARs comprise a transmembrane domain. The transmembrane domain of asubject CAR is a region that is capable of spanning the plasma membraneof a cell (e.g., an immune cell or precursor thereof). The transmembranedomain is for insertion into a cell membrane, e.g., a eukaryotic cellmembrane. In some embodiments, the transmembrane domain connects theextracellular/antigen binding domain and the intracellular domain of aCAR.

In some embodiments, the transmembrane domain is naturally associatedwith one or more of the domains in the CAR. In some embodiments, thetransmembrane domain can be selected or modified by one or more aminoacid substitutions to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins, to minimizeinteractions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein, e.g., a Type Itransmembrane protein. Where the source is synthetic, the transmembranedomain may be any artificial sequence that facilitates insertion of theCAR into a cell membrane, e.g., an artificial hydrophobic sequence.Examples of the transmembrane domains include, without limitation,transmembrane domains derived from (i.e. comprise at least thetransmembrane region(s) of) the alpha or beta chain of the T cellreceptor, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD7, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB),CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, or TLR9. In some embodiments, the transmembrane domainmay be synthetic, in which case it will comprise predominantlyhydrophobic residues such as leucine and valine. In some embodiments, atriplet of phenylalanine, tryptophan and valine are at each end of asynthetic transmembrane domain.

The transmembrane domains described herein can be combined with any ofthe antigen binding domains described herein, any of the intracellulardomains described herein, or any of the other domains described hereinthat may be included in a subject CAR.

In some embodiments, the transmembrane domain further comprises a hingeregion. Accordingly, in some embodiments, a subject CAR may also includea hinge region. The hinge region of the CAR is a hydrophilic regionwhich is located between the antigen binding domain and thetransmembrane domain. The hinge region may include a domain selectedfrom Fc fragments of antibodies, hinge regions of antibodies, CH2regions of antibodies, CH3 regions of antibodies, artificial hingesequences or combinations thereof. Examples of hinge regions include,without limitation, a CD8a hinge, artificial hinges made of polypeptideswhich may be as small as, three glycines (Gly), as well as CH1 and CH3domains of IgGs (such as human IgG4).

In some embodiments, a subject CAR of the present disclosure includes ahinge region that connects the antigen binding domain with thetransmembrane domain, which, in turn, connects to the intracellulardomain. The hinge region can be capable of supporting the antigenbinding domain to recognize and bind to the target antigen on the targetcells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2):125-135). In some embodiments, the hinge region is a flexible domain.Without being bound to any particular theory, the hinge region can allowthe antigen binding domain to have a structure to optimally recognizethe specific structure and density of the target antigens on a cell suchas tumor cell (Hudecek et al., supra). The flexibility of the hingeregion can permit the hinge region to adopt many differentconformations.

In some embodiments, the hinge region is an immunoglobulin heavy chainhinge region. In some embodiments, the hinge region is a hinge regionpolypeptide derived from a receptor (e.g., a CD8-derived hinge region).

The hinge region can have a length of from about 4 amino acids to about50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aato about 15 aa, from about 15 aa to about 20 aa, from about 20 aa toabout 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about40 aa, or from about 40 aa to about 50 aa. In some embodiments, thehinge region can have a length of greater than 5 aa, greater than 10 aa,greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa,greater than 50 aa, greater than 55 aa, or more.

Suitable hinge regions can be readily selected and can be of any of anumber of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acidsto 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitablehinge regions can have a length of greater than 20 amino acids (e.g.,30, 40, 50, 60 or more amino acids).

For example, hinge regions can include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO:1) and (GGGS)_(n) (SEQ ID NO: 2), where each n is,independently, an integer of at least one or 1-5), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers known inthe art. Glycine and glycine-serine polymers can be used. Glycinepolymers can also be used.

In some embodiments, the hinge region is an immunoglobulin heavy chainhinge region. Immunoglobulin hinge region amino acid sequences are knownin the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990)87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4):1779-1789. The hinge region can comprise an amino acid sequence of ahuman IgG1, IgG2, IgG3, or IgG4, hinge region. In some embodiments, thehinge region can include one or more amino acid substitutions and/orinsertions and/or deletions compared to a wild-type(naturally-occurring) hinge region.

In some embodiments, the hinge region can comprise an amino acidsequence derived from human CD8, or a variant thereof.

Intracellular Signaling Domain

A subject CAR also includes an intracellular signaling domain. The terms“intracellular signaling domain” and “intracellular domain” are usedinterchangeably herein. The intracellular signaling domain of the CAR isresponsible for activation of at least one of the effector functions ofthe cell in which the CAR is expressed (e.g., immune cell). Theintracellular signaling domain transduces the effector function signaland directs the cell (e.g., immune cell) to perform its specializedfunction, e.g., harming and/or destroying a target cell.

Examples of an intracellular domain include, but are not limited to, thecytoplasmic portion of a surface receptor, co-stimulatory molecule, andany molecule that acts in concert to initiate signal transduction in theT cell, as well as any derivative or variant of these elements and anysynthetic sequence that has the same functional capability.

Examples of intracellular signaling domains include, without limitation,the ζ chain of the T cell receptor complex or any of its homologs, e.g.,η chain, FcsRIγ and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc.,human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosinekinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn,etc.), and other molecules involved in T cell transduction, such as CD2,CD5 and CD28. In some embodiments, the intracellular signaling domainmay be human CD3 zeta chain, FcγRIII, FcsRI, cytoplasmic tails of Fcreceptors, an immunoreceptor tyrosine-based activation motif (ITAM)bearing cytoplasmic receptors, and combinations thereof.

In some embodiments, the intracellular signaling domain of the CARincludes any portion of one or more co-stimulatory molecules, such as atleast one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB,PD-1, any derivative or variant thereof, any synthetic sequence thereofthat has the same functional capability, and any combination thereof.

Other examples of the intracellular domain include a fragment or domainfrom one or more molecules or receptors including, but not limited to,TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon RIb), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40,CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory moleculesdescribed herein, any derivative, variant, or fragment thereof, anysynthetic sequence of a co-stimulatory molecule that has the samefunctional capability, and any combination thereof.

Additional examples of intracellular domains include, withoutlimitation, intracellular signaling domains of several types of variousother immune signaling receptors, including, but not limited to, first,second, and third generation T cell signaling proteins including CD3, B7family costimulatory, and Tumor Necrosis Factor Receptor (TNFR)superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol.(2015) 33(6): 651-653). Additionally, intracellular signaling domainsmay include signaling domains used by NK and NKT cells (see, e.g.,Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signalingdomains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012)189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol.(2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

Intracellular signaling domains of a CAR can include any desiredsignaling domain that provides a distinct signal (e.g., increasedproduction of one or more cytokines by the cell; change in transcriptionof a target gene; change in activity of a protein; change in cellbehavior, e.g., cell death; cellular proliferation; cellulardifferentiation; cell survival; modulation of cellular signalingresponses; etc.) in response to activation of the CAR (i.e., activatedby antigen and dimerizing agent). In some embodiments, the intracellularsignaling domain includes at least one (e.g., one, two, three, four,five, six, etc.) ITAM motifs as described below. In some embodiments,the intracellular signaling domain includes DAP10/CD28 type signalingchains. In some embodiments, the intracellular signaling domain is notcovalently attached to the membrane bound CAR, but is instead diffusedin the cytoplasm.

Intracellular signaling domains can also include immunoreceptortyrosine-based activation motif (ITAM)-containing intracellularsignaling polypeptides. In some embodiments, an ITAM motif is repeatedtwice in an intracellular signaling domain, where the first and secondinstances of the ITAM motif are separated from one another by 6 to 8amino acids. In some embodiments, the intracellular signaling domain ofa subject CAR comprises 3 ITAM motifs.

In some embodiments, intracellular signaling domains includes thesignaling domains of human immunoglobulin receptors that containimmunoreceptor tyrosine based activation motifs (ITAMs) such as, but notlimited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5(see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

A suitable intracellular signaling domain can also be an ITAMmotif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable intracellular signalingdomain can be an ITAM motif-containing domain from any ITAMmotif-containing protein. Thus, a suitable intracellular signalingdomain need not contain the entire sequence of the entire protein fromwhich it is derived. Examples of suitable ITAM motif-containingpolypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilonreceptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associatedprotein alpha chain).

In some embodiments, the intracellular signaling domain is derived fromDAP12 (also known as TYROBP; TYRO protein tyrosine kinase bindingprotein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associatedprotein; TYRO protein tyrosine kinase-binding protein; killer activatingreceptor associated protein; killer-activating receptor-associatedprotein; etc.). In one embodiment, the intracellular signaling domain isderived from FCER1G (also known as FCRG; Fc epsilon receptor I gammachain; Fc receptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRlgamma; high affinity immunoglobulin epsilon receptor subunit gamma;immunoglobulin E receptor, high affinity, gamma chain; etc.). In someembodiments, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA;T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, deltapolypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 deltachain; T-cell surface glycoprotein CD3 delta chain; etc.). In someembodiments, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 epsilon chain (also known as CD3e, T-cellsurface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In someembodiments, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 gamma chain (also known as CD3G, T-cellreceptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3complex), etc.). In some embodiments, the intracellular signaling domainis derived from T-cell surface glycoprotein CD3 zeta chain (also knownas CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q,T3Z, TCRZ, etc.). In some embodiments, the intracellular signalingdomain is derived from CD79A (also known as B-cell antigen receptorcomplex-associated protein alpha chain; CD79a antigen(immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha;membrane-bound immunoglobulin-associated protein; surface IgM-associatedprotein; etc.). In some embodiments, an intracellular signaling domainsuitable for use in a CAR of the present disclosure includes aDAP10/CD28 type signaling chain. In some embodiments, an intracellularsignaling domain suitable for use in a CAR of the present disclosureincludes a ZAP70 polypeptide. In some embodiments, the intracellularsignaling domain includes a cytoplasmic signaling domain of TCR zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, or CD66d. In some embodiments, the intracellular signalingdomain in the CAR includes a cytoplasmic signaling domain of human CD3zeta.

While usually the entire intracellular signaling domain can be employed,in many cases it is not necessary to use the entire chain. To the extentthat a truncated portion of the intracellular signaling domain is used,such truncated portion may be used in place of the intact chain as longas it transduces the effector function signal. The intracellularsignaling domain includes any truncated portion of the intracellularsignaling domain sufficient to transduce the effector function signal.

The intracellular signaling domains described herein can be combinedwith any of the antigen binding domains described herein, any of thetransmembrane domains described herein, or any of the other domainsdescribed herein that may be included in the CAR.

In Vivo CAR T

The present disclosure provides methods for producing or generating acell comprising a protein or polypeptide or polypeptide of interest, ina subject in vivo. In some embodiments, the methods compriseadministering the engineered viral particle(s), such as those providedfor herein. In some embodiments, the engineered viral particle comprisesan engineered envelope harboring a mutated fusion protein, a chimericgag protein, and an engineered targeting moiety for binding to a targetcell, wherein the mutated fusion protein does not bind to its naturalreceptor; and a nucleic acid encoding a polypeptide of interest. In someembodiments, the engineered viral particle comprises an engineeredenvelope harboring a mutated fusion protein and an engineered targetingmoiety for binding to a target cell, wherein the mutated fusion proteindoes not bind to its natural receptor; and a nucleic acid moleculeencoding a polypeptide of interest.

In some embodiments, the methods comprise administering a first viralparticle and a second viral particle. The first viral particle comprisesa chimeric gag protein, a first targeting moiety that binds to a firsttarget on a cell and a first nucleic acid molecule that encodes a firstportion of a protein or a polypeptide of interest. The second viralparticle comprises a chimeric gag protein, a second targeting moietythat binds to a second target on the cell and a second nucleic acidmolecule that encodes a second portion of the protein or the polypeptideof interest. When expressed in the cell, the first and second portionsof the protein or the polypeptide bind together, or interact with oneanother, to form a complete protein or polypeptide in the cell in thesubject.

In certain embodiments, the method further comprises administering adimerizing agent to the subject to form the protein or polypeptide inthe subject. Any dimerizing agent, as discussed in detail elsewhereherein, may be used. In certain embodiments, administering thedimerizing agent is required to form the complete protein, such as aCAR. The use of the dimerizing agent can provide an additional layer ofcontrol of the method and function of the protein in vivo.

In certain embodiments, the first portion and the second portion of theprotein or the polypeptide comprise an intein domain and the inteindomain is excised to link the first and second portion to form thecomplete protein or polypeptide.

In certain embodiments, the cell is a T cell, a CD4+ T cell, a CD8+ Tcell, a NK cell, an alpha-beta T cell, a gamma-delta T cell, a lymphoidprogenitor cell, a hematopoietic stem cell, a myeloid cell, a monocyte,a macrophage, a central memory T cell, a naïve T cells, an activated Tcell, a regulatory T Cell (Treg), or a CD8+ naïve/central memory cell(CD8+ CCR7+) and CD4+ naïve/central memory cell (CD4+CCR7+). In someembodiments, the cell is a CD4+CD8+ cell. These cell types are not to belimiting and as any type of cell can be generated using the methods andcompositions provided herein.

In certain embodiments, the polypeptide of interest is a chimericantigen receptor (CAR). Non-limiting examples of CARs are providedherein, but these are for illustrative purposes only and any type of CARcould be expressed in vivo using the compositions and methods providedherein.

In certain embodiments, the polypeptide of interest is a hemoglobin betachain.

In certain embodiments, the viral vectors or particles are administeredto the subject by injecting the vectors into the peripheral blood of thesubject. In certain embodiments, the viral vectors or particles areadministered to the subject by injecting the vectors into a lymph nodein the subject.

In some embodiments, methods of producing CART cells directly in asubject are provided that do not, in some embodiments, involve, or use,the ex vivo production, expansion, or activation of T cells. In someembodiments, the methods comprise using a lentiviral particlepseudotyped with (a) mutated measles and/or (b) Nipah virusglycoproteins. In some embodiments, the lentiviral particle is modifiedso that it cannot infect a target cell, without a specific targetingdomain also being present on the surface of the particle. In this way,CD4 T cells (using an anti-CD4 targeting domain) or CD8 T cells (usingan anti-CD8 targeting domain) are transduced. The transgene carried bythe vectors encodes a CAR. T cells can be transduced in the circulationby injection of the vectors into the peripheral blood. Alternatively, Tcells resident in lymphatic organs can be transduced by, for example,injecting vectors directly into a T cell rich organ such as a lymphnode.

Methods of Treatment

Also provided herein are methods of treating a disease in a subject inneed thereof.

In some embodiments, the methods provided include, but are not limitedto, methods of treating a disease in a subject in need thereof,comprising administering to the subject the viral particle(s) providedherein to treat the disease.

In certain embodiments, the disease is a cancer. In addition, thecompositions provided for herein can be used in methods for thetreatment of any condition related to a cancer, such as a cell-mediatedimmune response against a tumor cell(s), where it is desirable to treator alleviate the disease. The types of cancers to be treated include,but are not limited to, carcinoma, blastoma, sarcoma, certain leukemiaor lymphoid malignancies, benign and malignant tumors, malignanciese.g., sarcomas, carcinomas, and melanomas. Other exemplary cancersinclude, but are not limited to, breast cancer, prostate cancer, ovariancancer, cervical cancer, skin cancer, pancreatic cancer, colorectalcancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia,lung cancer, thyroid cancer, and the like. The cancers may be non-solidtumors (such as hematological tumors) or solid tumors. Adulttumors/cancers and pediatric tumors/cancers are also included. In oneembodiment, the cancer is a hematological tumor. In one embodiment, thecancer is a carcinoma. In one embodiment, the cancer is a sarcoma. Inone embodiment, the cancer is a leukemia. In one embodiment the canceris a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, medullary thyroid carcinoma, papillary thyroidcarcinoma, pheochromocytomas sebaceous gland carcinoma, papillarycarcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,seminoma, bladder carcinoma, melanoma, CNS tumors (such as a glioma(such as brainstem glioma and mixed gliomas), glioblastoma (also knownas glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases).

Carcinomas that can be amenable to therapy by the methods disclosedherein include, but are not limited to, esophageal carcinoma,hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer),squamous cell carcinoma (various tissues), bladder carcinoma, includingtransitional cell carcinoma (a malignant neoplasm of the bladder),bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastriccarcinoma, lung carcinoma, including small cell carcinoma and non-smallcell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

In certain exemplary embodiments, the compositions provided herein canbe used in methods to treat a myeloma, or a condition related tomyeloma. Examples of myeloma or conditions related thereto include,without limitation, light chain myeloma, non-secretory myeloma,monoclonal gamopathy of undetermined significance (MGUS), plasmacytoma(e.g., solitary, multiple solitary, extramedullary plasmacytoma),amyloidosis, and multiple myeloma. In some embodiments, a methods oftreating multiple myeloma are provided. In some embodiments, themultiple myeloma is refractory myeloma. In some embodiments, themultiple myeloma is relapsed myeloma.

In certain exemplary embodiments, the in vivo modified immune cellsproduced using the vectors and compositions provided herein are used totreat a melanoma, or a condition related to melanoma. Examples ofmelanoma or conditions related thereto include, without limitation,superficial spreading melanoma, nodular melanoma, lentigo malignamelanoma, acral lentiginous melanoma, amelanotic melanoma, or melanomaof the skin (e.g., cutaneous, eye, vulva, vagina, rectum melanoma). Insome embodiments, the melanoma is cutaneous melanoma. In someembodiments, the melanoma is refractory melanoma. In some embodiments,the melanoma is relapsed melanoma.

In some embodiments, the vectors and compositions provided herein areused to treat a sarcoma, or a condition related to sarcoma. Examples ofsarcoma or conditions related thereto include, without limitation,angiosarcoma, chondrosarcoma, chordoma, endotheliosarcoma, Ewing'ssarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma,liposarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,mesothelioma, malignant peripheral nerve sheath tumor, myxosarcoma,osteogenic sarcoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma,synovioma, synovial sarcoma, and other soft tissue sarcomas. In someembodiments, the sarcoma is synovial sarcoma. In some embodiments, thesarcoma is liposarcoma such as myxoid/round cell liposarcoma,differentiated/dedifferentiated liposarcoma, or pleomorphic liposarcoma.In some embodiments, the sarcoma is myxoid/round cell liposarcoma. Insome embodiments, the sarcoma is refractory sarcoma. In someembodiments, the sarcoma is relapsed sarcoma.

In certain embodiments, the compositions and vectors are used in methodsfor treating sickle cell disease. In some embodiments, the viral vectorsor particles are administered to a subject suffering from sickle celldisease, wherein the particles encode the hemoglobin beta chain. Whenexpressed in the cell, the hemoglobin beta chain is expressed andalleviates the symptoms of sickle cell disease to treat the disease.

In certain embodiments, the methods comprise administering a dimerizingagent to form the complete protein or polypeptide in the cell from theportions of the complete protein encoded by the plurality of thevectors.

In some embodiments, the subject has been treated with a therapeuticagent targeting the disease or condition, e.g. the tumor, prior toadministration of the composition or plurality of viral vectors. In someaspects, the subject is refractory or non-responsive to the othertherapeutic agent. In some embodiments, the subject has persistent orrelapsed disease, e.g., following treatment with another therapeuticintervention, including chemotherapy, radiation, and/or hematopoieticstem cell transplantation (HSCT), e.g., allogenic HSCT. In someembodiments, the administration effectively treats the subject despitethe subject having become resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeuticagent, and treatment with the therapeutic agent reduces disease burden.In some aspects, the subject is initially responsive to the therapeuticagent, but exhibits a relapse of the disease or condition over time. Insome embodiments, the subject has not relapsed. In some suchembodiments, the subject is determined to be at risk for relapse, suchas at a high risk of relapse, and thus the cells are administeredprophylactically, e.g., to reduce the likelihood of or prevent relapse.In some aspects, the subject has not received prior treatment withanother therapeutic agent.

The administration of the compositions may be carried out in anyconvenient manner known to those of skill in the art. For example, thecompositions may be administered to a subject by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patienttransarterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i.v.)injection, or intraperitoneally. In other instances, the compositions isinjected directly into a site of a local disease site in the subject, alymph node, an organ, a tumor, and the like.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the severity and course ofthe disease, whether the composition is administered for preventive ortherapeutic purposes, previous therapy, the subject's clinical historyand response to the treatment, and the discretion of the attendingphysician. The composition is, in some embodiments, suitablyadministered to the subject at one time or over a series of treatments.

In some embodiments, the composition is administered as part of acombination treatment, such as simultaneously with or sequentially with,in any order, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The composition(s), in some embodiments, is co-administered withone or more additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the composition is co-administered with anothertherapy sufficiently close in time such that the composition enhancesthe effect of one or more additional therapeutic agents, or vice versa.In some embodiments, the composition is administered prior to the one ormore additional therapeutic agents. In some embodiments, the compositionis administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agents includes a cytokine,such as IL-2, for example, to enhance persistence. In some embodiments,the methods comprise administration of a chemotherapeutic agent. In someembodiments, the methods do not comprise the administration of achemotherapeutic agent.

In certain embodiments, the compositions may be administered to asubject in combination with an immune checkpoint antibody (e.g., ananti-PD1, anti-CTLA-4, or anti-PDL1 antibody). For example, viralvectors may be administered in combination with an antibody or antibodyfragment targeting, for example, PD-1 (programmed death 1 protein).Examples of anti-PD-1 antibodies include, but are not limited to,pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known asMK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or anantigen-binding fragment thereof. In certain embodiments, thecompositions may be administered in combination with an anti-PD-L1antibody or antigen-binding fragment thereof. Examples of anti-PD-L1antibodies include, but are not limited to, BMS-936559, MPDL3280A(TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). Incertain embodiments, the composition may be administered in combinationwith an anti-CTLA-4 antibody or antigen-binding fragment thereof. Anexample of an anti-CTLA-4 antibody includes, but is not limited to,Ipilimumab (trade name Yervoy). Other types of immune checkpointmodulators may also be used including, but not limited to, smallmolecules, siRNA, miRNA, and CRISPR systems. Immune checkpointmodulators may be administered before, after, or concurrently with theviral vector. In certain embodiments, combination treatment comprisingan immune checkpoint modulator may increase the therapeutic efficacy ofa therapy comprising a composition as provided herein. The othertherapeutic can be administered simultaneously, before, or after thevectors provided herein are administered to the subject.

In certain embodiments, the subject is provided a secondary treatment.Secondary treatments include but are not limited to chemotherapy,radiation, surgery, and medications. In some embodiments, the subject isnot provided a secondary treatment.

In some embodiments, the methods are performed without a lymphodepletionstep, such as the administration of cyclophosphamide and/or fludarabine.

In some embodiments, the subject can be administered a conditioningtherapy after the administration of the vectors to kill certain immunecells that are not transduced with the CAR encoded by the plurality ofthe vectors. This can be done by including a selection marker that isencoded by the plurality of the vectors, such as described in U.S.Patent Publication No. 201909261223. In some embodiments, theconditioning therapy comprises administering an effective amount ofcyclophosphamide to the subject. In some embodiments, the conditioningtherapy comprises administering an effective amount of fludarabine tothe subject. In some embodiments, the conditioning therapy comprisesadministering an effective amount of a combination of cyclophosphamideand fludarabine to the subject.

In some embodiments, a specific dosage regimen of the present disclosureincludes a lymphodepletion step after the administration of thecomposition. In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide and/or fludarabine.

In some embodiments, the lymphodepletion step includes administration ofcyclophosphamide at a dose of between about 200 mg/m²/day and about 2000mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day). In anexemplary embodiment, the dose of cyclophosphamide is about 300mg/m²/day. In some embodiments, the lymphodepletion step includesadministration of fludarabine at a dose of between about 20 mg/m²/dayand about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day,or 60 mg/m²/day). In an exemplary embodiment, the dose of fludarabine isabout 30 mg/m²/day.

In some embodiment, the lymphodepletion step includes administration ofcyclophosphamide at a dose of between about 200 mg/m²/day and about 2000mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), andfludarabine at a dose of between about 20 mg/m²/day and about 900mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60mg/m²/day). In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide at a dose of about 300mg/m²/day, and fludarabine at a dose of about 30 mg/m²/day.

In an exemplary embodiment, the dosing of cyclophosphamide is 300mg/m²/day over three days, and the dosing of fludarabine is 30 mg/m²/dayover three days.

It is known in the art that one of the adverse effects of the use ofCART cells can be the onset of immune activation, known as cytokinerelease syndrome (CRS). CRS is immune activation resulting in elevatedinflammatory cytokines. CRS is a known on-target toxicity, developmentof which likely correlates with efficacy. Clinical and laboratorymeasures range from mild CRS (constitutional symptoms and/or grade-2organ toxicity) to severe CRS (sCRS; grade ≥3 organ toxicity, aggressiveclinical intervention, and/or potentially life threatening). Clinicalfeatures include: high fever, malaise, fatigue, myalgia, nausea,anorexia, tachycardia/hypotension, capillary leak, cardiac dysfunction,renal impairment, hepatic failure, and disseminated intravascularcoagulation. Dramatic elevations of cytokines includinginterferon-gamma, granulocyte macrophage colony-stimulating factor,IL-10, and IL-6 have been shown following CAR T-cell infusion. One CRSsignature is elevation of cytokines including IL-6 (severe elevation),IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations inclinically available markers of inflammation including ferritin andC-reactive protein (CRP) have also been observed to correlate with theCRS syndrome. The presence of CRS generally correlates with expansionand progressive immune activation of adoptively transferred cells. Ithas been demonstrated that the degree of CRS severity is dictated bydisease burden at the time of infusion as patients with high tumorburden experience a more sCRS.

Accordingly, in some embodiments, the methods comprise, following thediagnosis of CRS, appropriate CRS management strategies to mitigate thephysiological symptoms of uncontrolled inflammation without dampeningthe antitumor efficacy of the in vivo engineered cells (e.g., CAR Tcells). CRS management strategies are known in the art. For example,systemic corticosteroids may be administered to rapidly reverse symptomsof sCRS (e.g., grade 3 CRS) without compromising initial antitumorresponse.

In some embodiments, an anti-IL-6R antibody may be administered. Anexample of an anti-IL-6R antibody is the Food and DrugAdministration-approved monoclonal antibody tocilizumab, also known asatlizumab (marketed as Actemra, or RoActemra). Tocilizumab is ahumanized monoclonal antibody against the interleukin-6 receptor(IL-6R). Administration of tocilizumab has demonstrated near-immediatereversal of CRS.

CRS is generally managed based on the severity of the observed syndromeand interventions are tailored as such. CRS management decisions may bebased upon clinical signs and symptoms and response to interventions,not solely on laboratory values alone.

Mild to moderate cases generally are treated with symptom managementwith fluid therapy, non-steroidal anti-inflammatory drug (NSAID) andantihistamines as needed for adequate symptom relief. More severe casesinclude patients with any degree of hemodynamic instability; with anyhemodynamic instability, the administration of tocilizumab isrecommended. The first-line management of CRS may be tocilizumab, insome embodiments, at the labeled dose of 8 mg/kg IV over 60 minutes (notto exceed 800 mg/dose); tocilizumab can be repeated Q8 hours. Ifsuboptimal response to the first dose of tocilizumab, additional dosesof tocilizumab may be considered. Tocilizumab can be administered aloneor in combination with corticosteroid therapy. Patients with continuedor progressive CRS symptoms, inadequate clinical improvement in 12-18hours or poor response to tocilizumab, may be treated with high-dosecorticosteroid therapy, generally hydrocortisone 100 mg IV ormethylprednisolone 1-2 mg/kg. In patients with more severe hemodynamicinstability or more severe respiratory symptoms, patients may beadministered high-dose corticosteroid therapy early in the course of theCRS. CRS management guidance may be based on published standards (Lee etal. (2019) Biol Blood Marrow Transplant,doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev ClinOncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

Features consistent with Macrophage Activation Syndrome (MAS) orHemophagocytic lymphohistiocytosis (HLH) have been observed in patientstreated with CAR-T therapy (Henter, 2007), coincident with clinicalmanifestations of the CRS. MAS appears to be a reaction to immuneactivation that occurs from the CRS, and should therefore be considereda manifestation of CRS. MAS is similar to HLH (also a reaction to immunestimulation). The clinical syndrome of MAS is characterized by highgrade non-remitting fever, cytopenias affecting at least two of threelineages, and hepatosplenomegaly. It is associated with high serumferritin, soluble interleukin-2 receptor, and triglycerides, and adecrease of circulating natural killer (NK) activity.

In some embodiments, methods of treating cancer in a subject in needthereof are provided, the methods comprising administering to thesubject any of the compositions, such as the viral particle(s), providedherein. In some embodiments methods of treating cancer in a subject inneed thereof are provided, the methods comprising administering to thesubject a compostions generated by any one of the methods disclosedherein.

Pharmaceutical Compositions and Formulations

The compositions disclosed herein can comprise a pharmaceuticalcomposition, and for example include a pharmaceutically acceptablecarrier, and/or a pharmaceutical formulation.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. A “pharmaceutically acceptablecarrier” refers to an ingredient in a pharmaceutical formulation, otherthan an active ingredient, which is nontoxic to a subject. Apharmaceutically acceptable carrier includes, but is not limited to, abuffer, excipient, stabilizer, or preservative. In some aspects, thechoice of carrier is determined in part by the particular cell and/or bythe method of administration. Accordingly, there are a variety ofsuitable formulations. For example, the pharmaceutical composition cancontain preservatives. Suitable preservatives may include, for example,methylparaben, propylparaben, sodium benzoate, and benzalkoniumchloride. In some aspects, a mixture of two or more preservatives isused. The preservative or mixtures thereof are typically present in anamount of about 0.0001% to about 2% by weight of the total composition.Carriers are described, e.g., by Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being treated with thecomposition, preferably those with activities complementary to thecomposition, where the respective activities do not adversely affect oneanother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended. Thus, in someembodiments, the pharmaceutical composition further includes otherpharmaceutically active agents or drugs, such as chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.The pharmaceutical composition in some embodiments contains thecomposition in amounts effective to treat or prevent the disease orcondition, such as a therapeutically effective or prophylacticallyeffective amount. Therapeutic or prophylactic efficacy in someembodiments is monitored by periodic assessment of treated subjects. Thedesired dosage can be delivered by a single bolus administration of thecomposition, by multiple bolus administrations of the composition, or bycontinuous infusion administration of the composition. In someembodiments, the pharmaceutical composition does not include achemotherapeutic.

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, thecomposition is administered parenterally. The term “parenteral,” as usedherein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecomposition is administered to the subject using peripheral systemicdelivery by intravenous, intraperitoneal, or subcutaneous injection.Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyoi (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating thecomposition in a solvent, such as in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, dextrose, or the like. The compositions can contain auxiliarysubstances such as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, and/or colors, dependingupon the route of administration and the preparation desired. Standardtexts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

In some embodiments, the following embodiments are provided:

-   -   1. An engineered viral particle comprising        -   i. an engineered envelope comprising a mutated fusion            protein, a chimeric gag protein, and an engineered targeting            moiety for binding to a target cell, wherein the mutated            fusion protein does not bind to its natural receptor; and        -   ii. a nucleic acid encoding a polypeptide of interest.    -   2. The engineered viral particle of embodiment 1, wherein the        chimeric gag protein is xHIV gag protein.    -   3. The engineered viral particle of embodiments 1 or 2, wherein        the viral particle further comprises nucleotide sequence        encoding a SIV element, and/or a Pol protein.    -   4. The engineered viral particle of embodiment 2, wherein the        xHIV gag protein is encoded by the nucleic acid sequence as set        forth in SEQ ID NO: 5.    -   5. The engineered viral particle of embodiment 2, wherein the        xHIV gag protein is encoded by the nucleic acid sequence as set        forth in SEQ ID NO: 6.    -   6. The engineered viral particle of embodiment 3, wherein the        SIV element is encoded by the nucleic acid sequence as set forth        in SEQ ID NO: 7.    -   7. The engineered viral particle of embodiment 3, wherein the        Pol protein is encoded by the nucleic acid sequence as set forth        in SEQ ID NO: 8.    -   8. The engineered viral particle of embodiment 2, wherein the        xHIV gag protein comprises an amino acid sequence as set forth        in SEQ ID NO: 9.    -   9. The engineered viral particle of embodiment 3, wherein the        Pol protein comprises an amino acid sequence as set forth in SEQ        ID NO: 10.    -   10. The engineered viral particle of any one of embodiments 1-9,        wherein the targeting moiety is fused to the mutated fusion        protein.    -   11. The engineered viral particle of embodiments 1-10, wherein        the viral particle is a lentivirus pseudotyped with a measles        virus (MV) hemagglutinin (HA) protein or an MV fusion (F)        protein, a Nipah F protein, or a Nipah G protein, wherein the        MV-HA protein or the MV-F protein comprises a mutated binding        domain compared to its naturally occurring binding domain.    -   12. The engineered viral particle of embodiment 11, wherein the        targeting moiety is fused to the MV-HA protein the MV-F protein,        the Nipah F protein, or the Nipah G protein.    -   13. The engineered viral particle of any one of the preceeding        embodiments, wherein the targeting moiety is an antibody, a scFv        antibody, an antigen binding domain, a DARPIN, a VHH domain        antibody, a nanobody, single domain antibody, a FN3 domain, or        any combination thereof.    -   14. The engineered viral particle of any one of the preceeding        embodiments, wherein the targeting moiety is selected from the        group consisting of Stem Cell Factor protein (SCF, KIT-ligand,        KL, or steel factor) or a moiety that binds to cKit (CD117),        CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR        gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7,        CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.    -   15. The engineered viral particle of any one of the preceeding        embodiments, wherein the polypeptide of interest is a chimeric        antigen receptor (CAR) or a portion thereof    -   16. The engineered viral particle of embodiment 15, wherein the        chimeric antigen receptor comprises an extracellular domain,        transmembrane domain, and an intracellular signaling domain.    -   17. The engineered viral particle of embodiment 16, wherein the        extracellular domain binds to CD20, CD22, CD123, CD38, CD19,        BCMA, CD33, or CD79b.    -   18. The engineered viral particle of embodiment 1, wherein the        polypeptide of interest is a hemoglobin beta chain.    -   19. The engineered viral particle of any of the preceding        embodiments, wherein the viral particle is a pseudotyped        lentiviral vector, an adenovirus, or an adeno-associated virus.    -   20. The engineered viral particle of embodiment 19, wherein the        pseudotyped lentiviral vector is pseudotyped with a        morbillivirus or a henipavirus, such as a measles virus,        glycoprotein and/or a Nipah virus glycoprotein.    -   21. The engineered viral particle of embodiment 19, wherein the        pseudotyped vector is pseudotyped with a F protein or H protein        of a morbillivirus.    -   22. A method of in vivo gene delivery comprising administering        an engineered viral particle to a subject in need of delivery,        wherein the engineered viral particle comprises        -   i. an engineered envelope harboring a mutated fusion            protein, a chimeric gag protein and an engineered targeting            moiety for binding to a target cell, wherein the mutated            fusion protein does not bind to its natural receptor; and        -   ii. a nucleic acid encoding a polypeptide of interest        -   iii. wherein the administration of the engineered viral            particle induces an in vivo activity in the target cell            associated with the polypeptide of interest.    -   23. The method of embodiment 22, wherein the chimeric gag        protein is xHIV gag protein.    -   24. The method of embodiments 22 or 23, wherein the viral        particle further comprises a nucleotide sequence encoding a SIV        element, and/or a Pol protein.    -   25. The method of embodiment 23, wherein the xHIV gag protein is        encoded by the nucleic acid sequence as set forth in SEQ ID NO:        5.    -   26. The method of embodiment 23, wherein the xHIV gag protein is        encoded by the nucleic acid sequence as set forth in SEQ ID NO:        6.    -   27. The method of embodiment 24, wherein the SIV element is        encoded by the nucleic acid sequence as set forth in SEQ ID NO:        7.    -   28. The method of embodiment 24, wherein the Pol protein is        encoded by the nucleic acid sequence as set forth in SEQ ID NO:        8.    -   29. The method of embodiment 23, wherein the xHIV gag protein        comprises an amino acid sequence as set forth in SEQ ID NO: 9.    -   30. The method of embodiment 24, wherein the Pol protein        comprises an amino acid sequence as set forth in SEQ ID NO: 10.    -   31. The method of any one of embodiments 22-30, wherein the        target cell is a T cell, a CD4+ T cell, a CD8+ T cell, a NK        cell, an alpha-beta T cell, a gamma-delta T cell, a lymphoid        progenitor cell, a hematopoietic stem cell, a myeloid cell, a        monocyte, a macrophage, a central memory T cell, a naïve T cell,        an activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   32. A cell comprising the polypeptide of interest encoded for by        the engineered viral particle of any one of embodiments 1-21.    -   33. The cell of embodiment 32, wherein the cell is a T cell, a        CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T cell, a        gamma-delta T cell, a lymphoid progenitor cell, a hematopoietic        stem cell, a myeloid cell, a monocyte, a macrophage, a central        memory T cell, a naïve T cells, an activated T cell, or a        regulatory T Cell (Treg), or a T-Cell^(CD8+CCR7+).    -   34. A method of treating a disease in a subject, the method        comprising administering to the subject the engineered viral        particle of any one of embodiments 1-21, wherein the engineered        viral particle expresses the polypeptide of interest in a cell.    -   35. The method of embodiment 34, wherein the disease is cancer        or sickle cell disease.    -   36. The method of embodiments 34 or 35, wherein the cell is a T        cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T        cell, a gamma-delta T cell, a lymphoid progenitor cell, a        hematopoietic stem cell, a myeloid cell, a monocyte, a        macrophage, a central memory T cell, a naïve T cells, an        activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   37. A viral vector system comprising a first viral particle and        a second viral particle, wherein:        -   a.) the first viral particle comprises a chimeric gag            protein, a first targeting moiety that binds to a first            target on a cell and a first nucleic acid molecule that            encodes a first portion of a protein or a polypeptide of            interest;        -   b.) the second viral particle comprises a chimeric gag            protein, a second targeting moiety that binds to a second            target on the cell and a second nucleic acid molecule that            encodes a second portion of the protein or the polypeptide            of interest,        -   c.) wherein the first and second portions of the protein or            the polypeptide are capable of forming a complete protein or            polypeptide of interest inside the cell.    -   38. The viral vector system of embodiment 23, wherein the        chimeric gag protein is xHIV gag protein.    -   39. The viral vector system of embodiments 37 or 38, wherein the        viral particle further comprises nucleotide sequence encoding a        SIV element, and/or a Pol protein.    -   40. The viral vector system of embodiment 38, wherein the xHIV        gag protein is encoded by the nucleic acid sequence as set forth        in SEQ ID NO: 5.    -   41. The viral vector system of embodiment 38, wherein the xHIV        gag protein is encoded by the nucleic acid sequence as set forth        in SEQ ID NO: 6.    -   42. The viral vector system of embodiment 39, wherein the SIV        element is encoded by the nucleic acid sequence as set forth in        SEQ ID NO: 7.    -   43. The viral vector system of embodiment 39, wherein the Pol        protein is encoded by the nucleic acid sequence as set forth in        SEQ ID NO: 8.    -   44. The viral vector system of embodiment 38, wherein the xHIV        gag protein comprises an amino acid sequence as set forth in SEQ        ID NO: 9.    -   45. The viral vector system of embodiment 39, wherein the Pol        protein comprises an amino acid sequence as set forth in SEQ ID        NO: 10.    -   46. The viral vector system of any one of embodiments 37-45,        wherein the first target and the second target are different.    -   47. The viral vector system any one of embodiments 37-45,        wherein the protein or polypeptide of interest is a chimeric        antigen receptor.    -   48. The viral vector system of embodiment 47, wherein the        chimeric antigen receptor comprises an extracellular domain,        transmembrane domain, and an intracellular signaling domain.    -   49. The viral vector system of embodiment 48, wherein the        extracellular domain binds to CD20, CD22, CD123, CD38, CD19,        BCMA, CD33, or CD79b.    -   50. The viral vector system of any one of embodiments embodiment        37-45, wherein the protein or polypeptide is a hemoglobin beta        chain.    -   51. The viral vector system of any one of embodiments 37-50,        wherein the first targeting moiety and the second targeting        moiety are each independently selected from the group consisting        of a protein that binds to the first target, an antigen binding        domain, a DARPIN, and a FN3 domain, or any combination thereof.    -   52. The viral vector system of any one of embodiments 37-51,        wherein the first targeting moiety and the second targeting        moiety are each independently selected from the group consisting        of Stem Cell Factor protein (SCF, KIT-ligand, KL, or steel        factor) or a moiety that binds to cKit (CD117), CD4, CD8, CD3,        CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta,        CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2,        CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.    -   53. The viral vector system of embodiment 52, wherein the first        targeting moiety is selected from the group consisting of Stem        Cell Factor protein (SCF, KIT-ligand, KL, or steel factor) or a        moiety that binds to cKit (CD117), CD4, CD8, CD3, CD5, CD6, CD7,        CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34,        CD110, CD33, CD14, or CD68.    -   54. The viral vector system of embodiments 52 or 53, wherein the        second targeting moiety binds to CCR7, CD62L, CD25, CCR2, CCR3,        CCR4, CCR5, CCR6, CCR7, or CXCR3.    -   55. The viral vector system of any one of embodiments 37-51,        wherein the first target and the second target are each        independently selected from the group consisting of cKit        (CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta,        TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7,        CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.    -   56. The viral vector system of any one of embodiments 37-55,        wherein the first target is cKit (CD117), CD4, CD8, CD3, CD5,        CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10,        CD34, CD110, CD33, CD14, or CD68.    -   57. The viral vector system of any one of embodiments 37-56,        wherein the second target is CCR7, CD62L, CD25, CCR2, CCR3,        CCR4, CCR5, CCR6, CCR7, or CXCR3.    -   58. The viral vector system of any one of embodiments 37-57,        wherein the first viral vector is a pseudotyped viral vector.    -   59. The viral vector system of any one of embodiments 37-58,        wherein the second viral vector is a pseudotyped viral vector.    -   60. The viral vector system of any one of embodiments 37-59,        wherein the first and second viral vector are each,        independently, a pseudotyped lentiviral vector, an adenovirus,        or an adeno-associated virus.    -   61. The viral vector system of embodiment 60, wherein the        pseudotyped lentiviral vector is pseudotyped with a        morbillivirus, such as a measles virus, glycoprotein and/or a        Nipah virus glycoprotein.    -   62. The viral vector system of embodiment 60, wherein the first        and/or second viral vector is pseudotyped with a F protein or H        protein of a morbillivirus.    -   63. The viral vector system of any one of embodiments 37-62,        wherein the cell is a T cell, a CD4+ T cell, a CD8+ T cell, a NK        cell, an alpha-beta T cell, a gamma-delta T cell, a lymphoid        progenitor cell, a hematopoietic stem cell, a myeloid cell, a        monocyte, a macrophage, a central memory T cell, a naïve T cell,        an activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   64. The viral vector system of any one of embodiments 37-63,        wherein the first and second portions of the protein or        polypeptide form a complete protein in the presence of a        dimerizing agent.    -   65. The viral vector system of any one of embodiments 37-63,        wherein the protein or polypeptide is a chimeric antigen        receptor or a hemoglobin beta chain.    -   66. The viral vector system of embodiments 64 or 65, wherein the        dimerizing agent is rimiducid        ((1R)-3-(3,4-dimethoxyphenyl)-1-[3-({[2-(2-{3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2        S)-1-[(2        S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyloxy]propyl]phenoxy}acetamido)ethyl]carbamoyl}methoxy)phenyl]propyl        (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate        (AP1903)), erythropoietin, or rapamycin.    -   67. The viral vector system of any one of embodiments embodiment        37-63, wherein the first and second portions of the protein or        polypeptide can bind together through the excision of an intein        domain.    -   68. The viral vector system of any one of embodiments embodiment        37-45, wherein the first portion of the protein comprises an        extracellular domain of a chimeric antigen receptor and the        second portion of the protein comprises the transmembrane        domain, and an intracellular signaling domain.    -   69. A cell comprising the viral vector system of any one of        embodiments 37-68.    -   70. The cell of any one of embodiment 69, wherein the cell is a        T cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T        cell, a gamma-delta T cell, a lymphoid progenitor cell, a        hematopoietic stem cell, a myeloid cell, a monocyte, a        macrophage, a central memory T cell, a naïve T cells, an        activated T cell, or a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   71. A method of in vivo gene delivery comprising administering        to a subject in need of thereof a first viral particle and a        second viral particle, wherein:        -   a.) the first viral particle comprises a chimeric gag            protein, a first targeting moiety that binds to a first            target on a cell and a first nucleic acid molecule that            encodes a first portion of a protein or a polypeptide of            interest;        -   b.) the second viral particle comprises a chimeric gag            protein, a second targeting moiety that binds to a second            target on the cell and a second nucleic acid molecule that            encodes a second portion of the protein or the polypeptide            of interest,        -   c.) wherein the first and second portions of the protein or            the polypeptide are capable of forming a complete protein or            polypeptide of interest inside the cell.    -   72. The method of embodiment 71, wherein the chimeric gag        protein is xHIV gag protein.    -   73. The method of embodiment 71, further comprising        administering a dimerizing agent to form the protein or        polypeptide in the subject.    -   74. The method of embodiment 71, wherein the first portion and        the second portion comprise an intein domain and the intein        domain is excised to conjugate the first and second portion to        form the protein or polypeptide.    -   75. The method of embodiment 71, wherein the cell is a T cell, a        CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T cell, a        gamma-delta T cell, a lymphoid progenitor cell, a hematopoietic        stem cell, a myeloid cell, a monocyte, a macrophage, a central        memory T cell, a naïve T cells, an activated T cell, a        regulatory T Cell (Treg), or a CD8+ naïve/central memory cell        (CD8+ CCR7+) and CD4+ naïve/central memory cell (CD4+CCR7+).    -   76. The method of any one of embodiments 71-75, wherein the        protein or polypeptide is a chimeric antigen receptor.    -   77. The method of any one of embodiments 71-75, wherein the        protein or polypeptide is a hemoglobin beta chain.    -   78. The method of any one of embodiments 71-77, wherein the        first viral vector is a pseudotyped viral vector.    -   79. The method of any one of embodiments 71-78, wherein the        second viral vector is a pseudotyped viral vector.    -   80. The method of any one of embodiments 71-79, wherein the        first and second viral vector are each, independently, a        pseudotyped lentiviral vector, an adenovirus, or an        adeno-associated virus.    -   81. The method of embodiment 80, wherein the pseudotyped        lentiviral vector is pseudotyped with a morbillivirus or a        henipavirus, such as a measles virus, glycoprotein and/or a        Nipah virus glycoprotein.    -   82. The method of embodiment 80, wherein the first and/or second        viral vector is pseudotyped with a F protein or H protein of a        morbillivirus.    -   83. A method of treating a disease in a subject, the method        comprising administering to the subject a first viral particle        and a second viral particle, wherein:        -   a.) the first viral particle comprises a chimeric gag            protein, a first targeting moiety that binds to a first            target on a cell and a first nucleic acid molecule that            encodes a first portion of a protein or a polypeptide of            interest;        -   b.) the second viral particle comprises a chimeric gag            protein, a second targeting moiety that binds to a second            target on the cell and a second nucleic acid molecule that            encodes a second portion of the protein or the polypeptide            of interest,        -   c.) wherein the first and second portions of the protein or            the polypeptide are capable of forming a complete protein or            polypeptide of interest inside the cell.    -   84. The method of embodiment 83, wherein the disease is cancer        or sickle cell disease.    -   85. The method of embodiments 83 or 84, further comprising        administering a dimerizing agent to form the protein or        polypeptide in the cell.    -   86. The method of embodiments 83 or 84, wherein the first        portion and the second portion comprise an intein domain and the        intein domain is excised to conjugate the first and second        portion to form the protein or polypeptide.    -   87. The method of any one of embodiments 83-86, wherein the cell        is a T cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an        alpha-beta T cell, a gamma-delta T cell, a lymphoid progenitor        cell, a hematopoietic stem cell, a myeloid cell, a monocyte, a        macrophage, a central memory T cell, a naïve T cells, an        activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   88. The method of any one of embodiments 83-87, wherein the        protein or polypeptide is a chimeric antigen receptor.    -   89. The method of any one of embodiments 83-87, wherein the        protein or polypeptide is a hemoglobin beta chain.    -   90. The method of any one of embodiments 83-89, wherein the        first viral vector is a pseudotyped viral vector.    -   91. The method of any one of embodiments 83-90, wherein the        second viral vector is a pseudotyped viral vector.    -   92. The method of any one of embodiments 83-91, wherein the        first and second viral vector are each, independently, a        pseudotyped lentiviral vector, an adenovirus, or an        adeno-associated virus.    -   93. The method of embodiment 92, wherein the pseudotyped        lentiviral vector is pseudotyped with a morbillivirus, such as a        measles virus, glycoprotein and/or a Nipah virus glycoprotein.    -   94. The method of embodiment 93, wherein the first and/or second        viral vector is pseudotyped with a F protein or H protein of a        morbillivirus.    -   95. An engineered viral particle comprising an engineered        envelope comprising a mutated fusion protein and an engineered        targeting moiety for binding to a target cell, wherein the        mutated fusion protein does not bind to its natural receptor;        and a nucleic acid molecule encoding a polypeptide of interest.    -   96. The engineered viral particle of embodiment 95, wherein the        targeting moiety is fused to the mutated fusion protein.    -   97. The engineered viral particle of embodiments 95 or 96,        wherein the viral particle is a lentivirus pseudotyped with a        measles virus (MV) hemagglutinin (HA) protein or an MV        fusion (F) protein or a Nipah virus F or G protein,        -   a.) wherein the MV-HA protein, the MV-F protein, the Nipah            virus F, or the Nipah virus G protein comprises a mutated            binding domain compared to its naturally occurring receptor.    -   98. The engineered viral particle of embodiment 97, wherein the        targeting moiety is fused to the MV-HA protein, the MV-F        protein, the Nipah virus F, or the Nipah virus G protein.    -   99. The engineered viral particle of any one of embodiments        95-98, wherein the targeting moiety is a scFv, an antigen        binding domain, a DARPIN, a FN3 domain, a VHH, or any        combination thereof.    -   100. The engineered viral particle of any one embodiments 95-99,        wherein the targeting moiety is selected from the group        consisting of Stem Cell Factor protein (SCF, KIT-ligand, KL, or        steel factor) or a moiety that binds to cKit (CD117), CD4, CD8,        CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR        delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25,        CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.    -   101. The engineered viral particle of any one embodiments        95-100, wherein the polypeptide of interest is a chimeric        antigen receptor (CAR) or a portion thereof    -   102. The engineered viral particle of embodiment 101, wherein        the chimeric antigen receptor comprises an extracellular domain,        transmembrane domain, and an intracellular signaling domain.    -   103. The engineered viral particle of embodiment 102, wherein        the extracellular domain binds to CD20, CD22, CD123, CD38, CD19,        BCMA, CD33, or CD79b.    -   104. The engineered viral particle of embodiment 95, wherein the        polypeptide of interest is a hemoglobin beta chain.    -   105. The engineered viral particle of any one of embodiments        95-104, wherein the viral particle is a pseudotyped lentiviral        vector, an adenovirus, or an adeno-associated virus.    -   106. The engineered viral particle of embodiment 105, wherein        the pseudotyped lentiviral vector is pseudotyped with a        morbillivirus or henipavirus, such as a measles virus,        glycoprotein and/or a Nipah virus glycoprotein.    -   107. The engineered viral particle of embodiment 106, wherein        the pseudotyped vector is pseudotyped with a F protein and/or H        protein of a morbillivirus or a F protein and/or a G protein of        a henipavirus.    -   108. A method of in vivo gene delivery comprising administering        an engineered viral particle to a subject in need of delivery,        wherein the engineered viral particle is a particle of any one        of embodiments 95-108, wherein the administration of the        engineered viral particle induces an in vivo activity in a        target cell associated with the polypeptide of interest.    -   109. The method of embodiment 108, wherein the target cell is a        T cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T        cell, a gamma-delta T cell, a lymphoid progenitor cell, a        hematopoietic stem cell, a myeloid cell, a monocyte, a        macrophage, a central memory T cell, a naïve T cell, an        activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   110. A cell comprising the polypeptide of interest encoded for        by the engineered viral particle of any one of embodiments        95-108.    -   111. The cell of embodiment 110, wherein the cell is a T cell, a        CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T cell, a        gamma-delta T cell, a lymphoid progenitor cell, a hematopoietic        stem cell, a myeloid cell, a monocyte, a macrophage, a central        memory T cell, a naïve T cells, an activated T cell, or a        regulatory T Cell (Treg), or a T-Cell^(CD8+CCR7+).    -   112. A method of treating a disease in a subject, the method        comprising administering to the subject the engineered viral        particle of any one of embodiments 95-108, wherein the        engineered viral particle expresses the polypeptide of interest        in a cell.    -   113. The method of embodiment 112, wherein the disease is cancer        or sickle cell disease.    -   114. The method of embodiments 112 or 113, wherein the cell is a        T cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T        cell, a gamma-delta T cell, a lymphoid progenitor cell, a        hematopoietic stem cell, a myeloid cell, a monocyte, a        macrophage, a central memory T cell, a naïve T cells, an        activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   115. An engineered viral particle comprising:        -   i. an engineered envelope comprising a polypeptide having            the amino acid sequence of SEQ ID NO: 9;        -   ii. a heterologous polypeptide targeting moiety for binding            to a target cell; and        -   iii. a nucleic acid molecule encoding a heterologous            polypeptide of interest.    -   116. The engineered viral particle of embodiment 115, wherein        the targeting moiety is fused to a mutated fusion protein        present on the surface of the engineered viral particle.    -   117. The engineered viral particle of embodiment 115, wherein        the viral particle is a lentivirus pseudotyped with:        -   a) a measles virus (MV) hemagglutinin (HA) protein and/or an            MV fusion (F) protein and wherein the MV-HA protein or the            MV-F protein comprises a mutation or a mutated binding            domain compared to its naturally occurring protein; or        -   b) a Nipah virus F protein and/or a Nipah virus G protein            and wherein the a Nipah virus F protein and/or a Nipah virus            G protein comprises a mutation or a mutated binding domain            compared to its naturally occurring protein    -   118. The engineered viral particle of embodiment 115, wherein        the targeting moiety is fused to the MV-HA protein and/or the        MV-F protein or the Nipah virus F protein and/or a Nipah virus G        protein.    -   119. The engineered viral particle of embodiment 115, wherein        the targeting moiety is a scFv, an antigen binding domain, a        DARPIN, a VHH, or a FN3 domain.    -   120. The engineered viral particle of embodiment 115, wherein        the targeting moiety binds to protein selected from the group        consisting of Stem Cell Factor protein (SCF, KIT-ligand, KL, or        steel factor) or a moiety that binds to cKit (CDl 17), CD4, CD8,        CD3, CD3D, CD3E, CD3G, CD3Z, CD5, CD6, CD7, CD2, TCR alpha, TCR        beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68,        CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and        CXCR3.    -   121. The engineered viral particle of embodiment 115, wherein        the heterologous polypeptide of interest is a chimeric antigen        receptor (CAR).    -   122. The engineered viral particle of embodiment 121, wherein        the chimeric antigen receptor comprises an extracellular domain,        transmembrane domain, and an intracellular signaling domain.    -   123. The engineered viral particle of embodiment 122, wherein        the extracellular domain binds to CD20, CD22, CD123, CD38, CD19,        BCMA, CD33, or CD79b.    -   124. The engineered viral particle of embodiment 115, wherein        the heterologous polypeptide of interest is a hemoglobin beta        chain.    -   125. The engineered viral particle of embodiment 115, wherein        the viral particle is a pseudotyped lentiviral vector, an        adenovirus, or an adeno-associated virus.    -   126. The engineered viral particle of embodiment 125, wherein        the pseudotyped lentiviral vector is pseudotyped with a        morbillivirus or a henipavirus.    -   127. The engineered viral particle of embodiment 126, wherein        the morbillivirus is a measles virus.    -   128. The engineered viral particle of embodiment 126, wherein        the henipavirus is a Nipah virus.    -   129. The engineered viral particle of embodiment 125, wherein        the pseudotyped lentiviral vector comprises a morbillivirus F        protein and/or H protein.    -   130. The engineered viral particle of embodiment 129, wherein        the morbillivirus F protein and/or H protein is a measles F        protein and/or H protein.    -   131. The engineered viral particle of embodiment 130, wherein        the measles F protein comprises the amino acid sequence of SEQ        ID NO: 11 and the measles H protein comprises the amino acid        sequence of SEQ ID NO: 12.    -   132. The engineered viral particle of embodiment 125, wherein        the pseudotyped lentiviral vector comprises a henipavirus F        protein and/or G protein.    -   133. The engineered viral particle of embodiment 132, wherein        the henipavirus G protein is a Nipah G protein and the        henipavirus F protein is a Nipah F protein    -   134. The engineered viral particle of embodiment 133, wherein        the Nipah F protein comprises the amino acid sequence of SEQ ID        NO: 13 and the Nipah G protein comprises the amino acid sequence        of SEQ ID NO: 14.    -   135. A method of delivering a nucleic acid molecule encoding a        heterologous protein of interest to a cell, the method        comprising contacting the engineered viral particle of        embodiment 115 to a cell, thereby delivering the nucleic acid        molecule encoding the heterologous protein of interest to the        cell.    -   136. The method of embodiment 135, wherein the contacting        comprises administering the engineered viral particle of        embodiment 1 to a subject to deliver the nucleic acid molecule        encoding the heterologous protein of interest to a cell in vivo.    -   137. The method of embodiment 135, wherein the cell is contacted        with the engineered viral particle of embodiment ex vivo.    -   138. A viral vector system comprising a first viral particle and        a second viral particle, wherein:        -   a.) the first viral particle comprises a first targeting            moiety that binds to a first target on a cell and a first            nucleic acid molecule that encodes a first portion of a            protein or a polypeptide of interest;        -   b.) the second viral particle comprises a second targeting            moiety that binds to a second target on the cell and a            second nucleic acid molecule that encodes a second portion            of the protein or the polypeptide of interest,        -   wherein the first and second portions of the protein or the            polypeptide are capable of forming a complete protein or            polypeptide of interest inside the cell.    -   139. The viral vector system of embodiment 138, wherein the        first target and the second target are different.    -   140. The viral vector system of embodiment 138, wherein the        protein or polypeptide of interest is a chimeric antigen        receptor.    -   141. The viral vector system of embodiment 139, wherein the        chimeric antigen receptor comprises an extracellular domain,        transmembrane domain, and an intracellular signaling domain.    -   142. The viral vector system of embodiment 141, wherein the        extracellular domain binds to CD20, CD22, CD123, CD38, CD19,        BCMA, CD33, or CD79b.    -   143. The viral vector system of embodiment 138, wherein the        protein or polypeptide is a hemoglobin beta chain.    -   144. The viral vector system of any one of embodiments 138-143,        wherein the first targeting moiety and the second targeting        moiety are each independently selected from the group consisting        of a protein that binds to the first target, an antigen binding        domain, a DARPIN, a VHH, a scFV, and a FN3 domain, or any        combination thereof.    -   145. The viral vector system of any one of embodiments 138-144,        wherein the first targeting moiety and the second targeting        moiety are each independently selected from the group consisting        of Stem Cell Factor protein (SCF, KIT-ligand, KL, or steel        factor) or a moiety that binds to cKit (CD117), CD4, CD8, CD3,        CD5, CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta,        CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2,        CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.    -   146. The viral vector system of embodiment 145, wherein the        first targeting moiety is selected from the group consisting of        Stem Cell Factor protein (SCF, KIT-ligand, KL, or steel factor)        or a moiety that binds to cKit (CD117), CD4, CD8, CD3, CD5, CD6,        CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34,        CD110, CD33, CD14, or CD68.    -   147. The viral vector system of embodiments 145 or 146, wherein        the second targeting moiety binds to CCR7, CD62L, CD25, CCR2,        CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3.    -   148. The viral vector system of any one of embodiments 138-145,        wherein the first target and the second target are each        independently selected from the group consisting of cKit        (CD117), CD4, CD8, CD3, CD5, CD6, CD7, CD2, TCR alpha, TCR beta,        TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7,        CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CXCR3.    -   149. The viral vector system of any one of embodiments 138-148,        wherein the first target is cKit (CD117), CD4, CD8, CD3, CD5,        CD6, CD7, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10,        CD34, CD110, CD33, CD14, or CD68.    -   150. The viral vector system of any one of embodiments 138-149,        wherein the second target is CCR7, CD62L, CD25, CCR2, CCR3,        CCR4, CCR5, CCR6, CCR7, or CXCR3.    -   151. The viral vector system of any one of embodiments 138-150,        wherein the first viral vector is a pseudotyped viral vector.    -   152. The viral vector system of any one of embodiments 138-151,        wherein the second viral vector is a pseudotyped viral vector.    -   153. The viral vector system of any one of embodiments 138-152,        wherein the first and second viral vector are each,        independently, a pseudotyped lentiviral vector, an adenovirus,        or an adeno-associated virus.    -   154. The viral vector system of embodiment 153, wherein the        pseudotyped lentiviral vector is pseudotyped with a        morbillivirus or henipavirus, such as a measles virus H protein        or glycoprotein and/or a Nipah virus F protein and/or G protein.    -   155. The viral vector system of embodiment 154, wherein the        first and/or second viral vector is pseudotyped with a F protein        or H protein of a morbillivirus.    -   156. The viral vector system of any one of embodiments 138-155,        wherein the cell is a T cell, a CD4+ T cell, a CD8+ T cell, a NK        cell, an alpha-beta T cell, a gamma-delta T cell, a lymphoid        progenitor cell, a hematopoietic stem cell, a myeloid cell, a        monocyte, a macrophage, a central memory T cell, a naïve T cell,        an activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   157. The viral vector system of any one of embodiments 138-156,        wherein the first and second portions of the protein or        polypeptide form a complete protein in the presence of a        dimerizing agent.    -   158. The viral vector system of any one of embodiments 138-157,        wherein the protein or polypeptide is a chimeric antigen        receptor or a hemoglobin beta chain.    -   159. The viral vector system of embodiments 157 or 158, wherein        the dimerizing agent is rimiducid        ((1R)-3-(3,4-dimethoxyphenyl)-1-[3-({[2-(2-{3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyloxy]propyl]phenoxy}acetamido)ethyl]carbamoyl}methoxy)phenyl]propyl        (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate        (AP1903)), erythropoietin, or rapamycin.    -   160. The viral vector system of any one of embodiments        embodiment 138-156, wherein the first and second portions of the        protein or polypeptide can bind together through the excision of        intein domain.    -   161. The viral vector system of embodiment 138, wherein the        first portion of the protein comprises an extracellular domain        of a chimeric antigen receptor and the second portion of the        protein comprises the transmembrane domain, and an intracellular        signaling domain.    -   162. A cell comprising a first nucleic acid molecule that        encodes a first portion of a chimeric antigen receptor and a        second nucleic acid molecule that encodes a second portion of        the chimeric antigen receptor, wherein the first portion of the        chimeric antigen receptor and the second portion of the chimeric        antigen receptor are capable of binding to one another to form a        complete chimeric antigen receptor in the presence of a        dimerizing agent or dimerizing domain.    -   163. The cell of embodiment 162, wherein the dimerizing agent is        rimiducid        ((1R)-3-(3,4-dimethoxyphenyl)-1-[3-({[2-(2-{3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2        S)-1-[(2        S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carbonyloxy]propyl]phenoxy}acetamido)ethyl]carbamoyl}methoxy)phenyl]propyl        (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate        (AP1903)), erythropoietin, or rapamycin.    -   164. The cell of embodiment 163, wherein the dimerizing domain        is an intein domain.    -   165. A cell comprising a first nucleic acid molecule that        encodes a first portion of a protein or polypeptide and a second        nucleic acid molecule that encodes a second portion of the        protein or the polypeptide, wherein the first and second        portions of the protein or the polypeptide can bind together to        form a complete protein or polypeptide.    -   166. The cell of embodiment 165, wherein the protein or        polypeptide is a hemoglobin beta chain.    -   167. The cell of embodiment 165, wherein the protein or        polypeptide is a heterologous chimeric antigen receptor, wherein        the heterologous chimeric antigen chimer receptor comprises a        first portion of the chimeric antigen receptor and a second        portion of the chimeric antigen receptor, wherein the first        portion and the second portion bind to one another to form the        chimeric antigen receptor through a dimerizing agent or        dimerizing domain, such as an intein domain.    -   168. The cell of any one of embodiments 165-167, wherein the        cell is a T cell, a CD4+ T cell, a CD8+ T cell, a NK cell, an        alpha-beta T cell, a gamma-delta T cell, a lymphoid progenitor        cell, a hematopoietic stem cell, a myeloid cell, a monocyte, a        macrophage, a central memory T cell, a naïve T cells, an        activated T cell, or a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   169. A method of in vivo or ex vivo gene delivery comprising        administering to a subject in need of delivery a first viral        particle and a second viral particle, wherein:        -   a.) the first viral particle comprises a first targeting            moiety that binds to a first target on a cell and a first            nucleic acid molecule that encodes a first portion of a            protein or polypeptide of interest;        -   b.) the second viral particle comprises a second targeting            moiety that binds to a second target on the cell and a            second nucleic acid molecule that encodes a second portion            of the protein or the polypeptide of interest,        -   wherein the first and second portions of the protein or the            polypeptide are capable of forming a complete protein or            polypeptide inside the cell.    -   170. The method of embodiment 169, further comprising        administering a dimerizing agent to form the protein or        polypeptide in the subject.    -   171. The method of embodiment 169, wherein the first portion and        the second portion comprise an intein domain and the intein        domain is excised to conjugate the first and second portion to        form the protein or polypeptide.    -   172. The method of embodiment 169, wherein the cell is a T cell,        a CD4+ T cell, a CD8+ T cell, a NK cell, an alpha-beta T cell, a        gamma-delta T cell, a lymphoid progenitor cell, a hematopoietic        stem cell, a myeloid cell, a monocyte, a macrophage, a central        memory T cell, a naïve T cells, an activated T cell, a        regulatory T Cell (Treg), or a CD8+ naïve/central memory cell        (CD8+ CCR7+) and CD4+ naïve/central memory cell (CD4+CCR7+).    -   173. The method of any one of embodiments 169-172, wherein the        protein or polypeptide is a chimeric antigen receptor.    -   174. The method of any one of embodiments 169-172 wherein the        protein or polypeptide is a hemoglobin beta chain.    -   175. The method of any one of embodiments 169-174, wherein the        first viral vector is a pseudotyped viral vector.    -   176. The method of any one of embodiments 169-175, wherein the        second viral vector is a pseudotyped viral vector.    -   177. The method of any one of embodiments 169-176, wherein the        first and second viral vector are each, independently, a        pseudotyped lentiviral vector, an adenovirus, or an        adeno-associated virus.    -   178. The method of embodiment 177, wherein the pseudotyped        lentiviral vector is pseudotyped with a morbillivirus or a        henipavirus, such as a measles virus, glycoprotein and/or a        Nipah virus glycoprotein.    -   179. The method of embodiment 178, wherein the first and/or        second viral vector is pseudotyped with a F protein or H protein        of a morbillivirus or a F protein or G protein of a henipavirus.    -   180. A method of treating a disease in a subject, the method        comprising administering to the subject a first viral particle        and a second viral particle, wherein:        -   a.) the first viral particle comprises a first targeting            moiety that binds to a first target on a cell and a first            nucleic acid molecule that encodes a first portion of a            protein or polypeptide of interest;        -   b.) the second viral particle comprises a second targeting            moiety that binds to a second target on the cell and a            second nucleic acid molecule that encodes a second portion            of the protein or the polypeptide of interest,        -   wherein the first and second portions of the protein or the            polypeptide are capable of forming a complete protein or            polypeptide in the cell to treat the disease.    -   181. The method of embodiment 180, wherein the disease is cancer        or sickle cell disease.    -   182. The method of embodiments 180 or 181, further comprising        administering a dimerizing agent to form the protein or        polypeptide in the cell.    -   183. The method of any one of embodiments 180-182, wherein the        first portion and the second portion comprise an intein domain        and the intein domain is excised to conjugate the first and        second portion to form the protein or polypeptide.    -   184. The method of embodiments any one of embodiments 180-183,        wherein the cell is a T cell, a CD4+ T cell, a CD8+ T cell, a NK        cell, an alpha-beta T cell, a gamma-delta T cell, a lymphoid        progenitor cell, a hematopoietic stem cell, a myeloid cell, a        monocyte, a macrophage, a central memory T cell, a naïve T        cells, an activated T cell, a regulatory T Cell (Treg), or a        T-Cell^(CD8+CCR7+).    -   185. The method of any one of embodiments 180-184, wherein the        protein or polypeptide is a chimeric antigen receptor.    -   186. The method of any one of embodiments 180-184 wherein the        protein or polypeptide is a hemoglobin beta chain.    -   187. The method of any one of embodiments 180-186, wherein the        first viral vector is a pseudotyped viral vector.    -   188. The method of any one of embodiments 180-187, wherein the        second viral vector is a pseudotyped viral vector.    -   189. The method of any one of embodiments 180-188, wherein the        first and second viral vector are each, independently, a        pseudotyped lentiviral vector, an adenovirus, or an        adeno-associated virus.    -   190. The method of embodiment 189, wherein the pseudotyped        lentiviral vector is pseudotyped with a morbillivirus, such as a        measles virus, glycoprotein and/or a Nipah virus glycoprotein.    -   191. The method of embodiment 190, wherein the first and/or        second viral vector is pseudotyped with a F protein and/or H        protein of a morbillivirus or a F protein and/or a G protein of        a henipavirus.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. It will be readily apparent to those skilled in the art thatother suitable modifications and adaptations of the methods describedherein may be made using suitable equivalents without departing from thescope of the embodiments disclosed herein. In addition, manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to the objective,spirit and scope of the present invention. All such modifications areintended to be within the scope of the claims appended hereto. Havingnow described certain embodiments in detail, the same will be moreclearly understood by reference to the following examples, which areincluded for purposes of illustration only and are not intended to belimiting.

EXAMPLES

The embodiments are now described with reference to the followingExamples. These Examples are provided for the purpose of illustrationonly, and the embodiments are not limited to these Examples, but ratherencompasses all variations that are evident as a result of the teachingsprovided herein.

Example 1 Pseudotyped Viral Vector System Containing Chimeric Gag

The present disclosure is, in part, based on the finding thatsubstituting xHIV gag protein, a chimeric gag protein from SIV, for theHIV gag protein found in standard pseudotyped lentiviral vectors,enhances transduction efficiency.

Improved measles virus (MV)-pseudotyped lentiviral vectors and viralvector systems were generated herein by replacing the HIV gag-polsequence (illustrated in the FIG. 1A) with a sequence encoding xHIV asset forth in SEQ ID NO: 5, which comprises xHIV gag protein, which is achimeric gag protein from SIV and HIV (FIG. 1B) (Uchida et al. J. Virol,October 2009, p. 9854-9862; Pavlakis, U.S. Pat. No. 8,076,100B2) (FIGS.1A-1B). The xHIV gag protein encoded by the polynucleotide sequence isprotein of SEQ ID NO: 9.

Activated PBMCs were transduced with a pseudotyped viral vector systemcontaining HIV gag-pol and truncated NGFR (gag-pol NGFR MV) (FIG. 2A andFIG. 2C) or a pseudotyped viral vector system containing the chimericxHIV and truncated NGFR (xHIV NGFR MV) (FIG. 2B and FIG. 2D). Resultsshowed that on day 4, CD4⁺ were transduced at a higher rate when usingthe pseudotyped viral vector system containing the chimeric xHIV (xHIVNGFR MV) compared to the vector system containing the HIV gag-pol(gag-pol NGFR MV) (FIGS. 2A-2B). Additionally, the xHIV vector displayedhigher transduction efficiency (FIGS. 2C and 2D).

Similar results were obtained from experiments wherein activated PBMCswere transduced with gag-pol GFP MV (FIG. 3A and FIG. 3C) or xHIV GFP MV(FIG. 3B and FIG. 3D). Results from day 4 showed a 3 fold increase inpotency with vectors containing chimeric gag (FIGS. 3A-3D).

Activated PBMCs were transduced with pseudotyped vectors containingchimeric antigen receptors that target CD19 (CAR-19), packaged usingeither the HIV gag-pol (gag-pol CAR-19 MV) (FIG. 4A and FIG. 4C) or thechimeric gag (xHIV CAR-19 MV) (FIG. 4B and FIG. 4D). Again, transductionefficiency was increased with the vectors containing the chimeric gag(FIGS. 4A-4B) and 4-5 times less virus was needed (FIGS. 4C-4D).

Results from day 12 also demonstrate increased transduction efficienciesin chimeric gag containing vectors (FIGS. 5A-5D, FIGS. 6A-6D, and FIGS.7A-7D) with higher dilutions displaying larger differences (FIG. 5B,FIG. 5D, FIG. 6B and FIG. 6D). Importantly, an increase in transductionefficiency on day 12 was demonstrated with the pseudotyped vectorscontaining CAR-19 and chimeric gag (FIGS. 7A-7D), while 10 times lessvolume was needed (FIG. 7C and FIG. 7D).

Prior to the present invention, the state of the art of gene transferinto T cells for cancer therapy included a variety of ex vivo approachesthat range from viral (e.g. lentiviral transduction) to non-viralapproaches (e.g. electroporation of transposon plasmids). Since theanti-tumor effect of CAR T cells depends upon their ability toproliferate, stable integration of the transgene is critical.Lentiviruses (LV) commonly used in gene delivery typically (i) lack thepathogenic features of the parental HIV; (ii) lack the ability togenerate further infectious particles, and (iii) stably infect a widevariety of cells (by replacing the HIV envelope protein with the Gglycoprotein from the vesicular stomatitis virus; VSVG). Susceptibilityof cells to infection with VSVG-pseudotyped LV requires expression ofthe LDL receptor. While activated T cells express the LDL receptor,resting lymphocytes do not and are therefore resistant to transductionwith standard VSVG-pseudotyped LV vectors. Thus, to date, geneticmodification of T cells has been carried out ex vivo because (i) onlythe cells of interest are exposed to the virus thus increasing safetyand reducing the amount of viral particles needed, and (ii) T cells canbe activated ex vivo using anti-CD3/CD28 beads or similar techniques inorder to enhance expression of the LDL receptor and improvetransduction.

Among the major challenges facing patients and physicians is theduration of the ex vivo manufacturing process, which is at least 17 daysand often much longer. Additional logistical issues that limit access,increase time-to-treatment, and generate costs include apheresisavailability, GMP suite availability in time for manufacturing, andrelease testing of each product which is thus treated as a new lot.

As disclosed herein, all these issues could be circumvented by in vivotransduction of T cells after systemic administration in the patientusing a specific, efficient viral system. It is contemplated that such aviral system has the following features: (i) viral particles are able tospecifically transduce the target cells of interest, (ii) transductionefficiency is high enough to lead to a sufficient number of T cellscarrying the transgene after in vivo administration, and (iii) thetransduced cells are functional.

To facilitate in vivo gene transfer, viral particles are engineered tobind a cell surface marker of choice for cell entry, rather than theirnatural receptors. The viral particles express a targeting ligand suchas a single-chain variable fragment (scFv) in order to accomplishspecific binding to the surface of the target cell. For example, LV ispseudotyped with measles virus hemagglutinin (HA) and fusion (F)proteins that were mutated to prevent binding to their natural receptorsCD46 and SLAM. In order to then confer specificity on the MV-pseudotypedLV, the HA gene is modified to express a targeting domain, such as ascFV against CD8 or CD4. In addition, the sequence encoding the HIV gagprotein in the pseudotyped lentiviral vector was replaced with achimeric SIV Gag protein, xHIV. The incorporation of the chimeric gaginto the pseudotyped lentiviral vector unexpectedly resulted inenhancing the transduction efficiency without loss of specificity.

Example 2 Erythropoietin Receptor (EpoR)-Based Binding and SignalingComponents

A schematic illustrating another embodiment of a signaling complex ofthe present disclosure is shown in FIGS. 8A-8B wherein the EpoRsignaling (e.g., EpoR 4-1BB-CD3 zeta (BBz) endodomain encoded bytransgene 1 from lentiviral vector 1 (LV 1)) and EpoR binding (e.g., theCAR-EpoR exodomain encoded by transgene 2 from lentiviral vector 2 (LV2)) components are individually expressed in a cell (e.g., T cell) usingdifferent lentiviral vectors for each (FIG. 8A). This exemplifiedEPOR-based system separates the antigen recognition (e.g., scFV CAR 19)and signaling functions of a CAR into two distinct polypeptides thatcontain the dimerization domains of EpoR that dimerize to providesignaling activity in the presence of the dimerizating agent EPO (FIG.8B). The expression of CAR-EpoR exodomain is shown in FIG. 9 and theexpression of EpoR BBz endodomain is shown in FIG. 10 .

Such a split vector application can be tailored such that the exodomainvector has a first targeting moiety that binds to a first target (e.g.,CD8) on a cell, whereas the endodomain vector has a second targetingmoiety that binds to a second target (e.g., CCR7) on the cell, or viceversa. In the schematic of such a concept illustrated in FIG. 11 , onlyCD8+CCR7+ cells can contain both the exo- and endo-domains, which can beactivated via the CAR and with administration of EPO.

Example 3 Specific Transduction of Human CD4 Cells in Blood or Liver ofHumanized Mice

Humanized mice were injected with a measles virus (MV-CD4) lentiviralvector targeting CD4 cells and comprising a GFP reporter, a vesicularstomatitis virus G-protein (VSVG) lentiviral vector, or PBS. At 7 daysafter IV administration, blood was extracted and subjected to analysisusing flow cytometry. The data, as provided in FIG. 12 , showed specifictransduction of human CD4 cells in blood of humanized mice with theMC-CD4 vector.

In the same experiment, livers were extracted 7 days following IVadministration of MV-CD4, VSVG, or PBS, sectioned and subjected toimmunohistochemical staining. The data, as provided in FIG. 13 , showedno positive staining against CD4 and GFP in animals injected with PBS,staining of Kupffer cells in animals injected with VSVG lentiviralvector, and staining of the lymphoid island in liver perenchyma ofanimals injected with MV-CD4. Kupffer cells are the resident macrophagesof the liver and primary target of VSVG pseudotyped lentiviral vectors.Positive staining of a lymphoid follicle in liver parenchyma of animalsinjected with MV-CD4 shows specific transduction of human CD4 cells inliver of humanized mice.

Example 4 Specific Transduction of Human CD4 Cells in Peritoneum ofHumanized Mice

Humanized mice were injected with MV-CD4 lentiviral vector comprising aNGFR reporter, a VSVG lentiviral vector, or PBS. Peritoneal cells wererecovered by lavage 7 days following IV administration of MV-CD4, VSVG,or PBS, and subjected to flow cytometry. The data, as provided in FIG.14 , showed expression of NGFR in human cells from the peritonealcavity. Accordingly, CD4+ T cells of humanized mice show specifictransduction with MV-CD4.

Example 5

Specific transduction of resting (non-activated) rhesus macaque CD4cells in vitro. PBMCs of rhesus macaque were transduced with a MVlentiviral comprising a NGFR reporter, a Nipah lentiviral vector (NV), aVSVG lentiviral vector, or PBS. Four days after transduction, restingNHP T cells were subjected to flow cytometry. The data, as provided inFIG. 15 , showed expression of NGFR and CD4 in cells transduced with MVor NV, but not VSVG or in untransduced cells. Accordingly, resting NHP Tcells transduced with MV or NV express CD4.

Example 6 Stimulated Human T Cells Transduced with the MV Anti-CD8DARPIN Vector Show Efficient and Specific Transgene Expression

Human T cells were stimulated using anti-CD3/CD28 beads, transduced withMV pseudotyped anti-CD8 DARPIN redirected vector made using xHIV gag andactivated with IL7 and IL15. Cells were subjected to flow cytometry onday 11. The data, as provided in FIG. 16 show the expected profile ofprotein expression at 3-fold dilutions of viral vector, with anexemplary plot shown in FIG. 17 . Accordingly, stimulated human T cellstransduced with the MV anti-CD8 DARPIN vector show efficient andspecific transgene expression.

Example 7 Anti-CD19 CAR MV-CD4, Anti-CD19 CAR NiV-CD4, and Anti-CD20 CARNiV-CD4 Vectors are Functional

Degranulation was assessed in Raji or Ramos CD19+CD20+ lymphoma celllines in response to anti-CD20 or anti-CD19 BBz CAR20 (VSVG), oranti-CD20 or anti-CD19 KIR MV or NV systems. Degranulation was measuredusing flow cytometry by calculating the percentage of CD107a+ cellswithin the population of CD4+ cells. The data, as provided in FIG. 18 ,showed specific degranulation of T cells transduced with VSVG, anti-CD19CAR MV-CD4, anti-CD19 CAR NiV-CD4, or anti-CD20 CAR NiV-CD4, but notanti-CD20-MV-CD4 upon exposure to both cell lines. Accordingly,anti-CD19 CAR MV-CD4, anti-CD19 CAR NiV-CD4, and anti-CD20 CAR NiV-CD4vectors increase degranulation of CD4+ cells.

Example 8 AntiCD20 KIR CAR MV are Functional, Albeit with SlowerKinetics than Anti-CD20 BBz CAR20 (VSVG)

T cells transduced with KIRCAR20, at efficiency of 17%, were incubatedwith luciferase-expressing Z138 lymphoma cells at 5:1, 2.5:1, 1.25:1,0.625:1, 0.3125:1, 0.15625:1, or 0.0781:1 E:T ratios. Cytotoxicity wasevaluated at 48 hours following transduction and defined as reduction inluciferase activity, and is shown in FIG. 19 . T cells transduced withthe KIRCAR20 system required higher E:T ratios to induce specific lysisthan the standard BBz-costimulated CAR system. Therefore, T cellstransduced with the KIRCAR20 system are functional but induce lessprompt killing than the standard BBz CAR system.

Example 9 Cells Transduced with the Virus Comprising the IL15/IL15RExpression Cassette Show Improved Proliferation

Proliferation of resting human T cells transduced with virusesincorporating an IL15/IL15R expression cassette was measured byanalyzing the cell concentration, and is shown in FIG. 20 . Cellstransduced with the virus incorporating an IL15/IL15R expression showed10-fold higher expansion of cells transduced to express the cytokineresponse cassette compared to controls. Accordingly, cells transducedwith the virus comprising the cytokine response cassette show improvedproliferation.

Example 10 MV Pseudotyped Lentiviral Vectors Transduce CD8 and CD3eCells

Human T cells were activated with beads and exposed to a CD8 or CD3etargeting MV-pseudotyped lentiviral vector. Flow cytometry data showedexpression of NGFR and CD8 or NGFR and CD3e. Accordingly, the MVpseudotyped lentiviral vector targets CD8 and CD3e cells.

Example 11 MV-Based Transduction is Highly Specific and Efficient InVivo

Human PBMC (10E6) were engrafted into NSG mice and intraperitoneallyinjected with 2.5×10⁶ TU CD4-specific MV vector, or VSVG vector. Micewere sacrificed at 2 days or 7 days following administration of thevirus. Transduction was measured in the peritoneum, spleen, blood, andmarrow. Mice injected with the MV vector showed on target transductionin all analyzed tissues or cells, as compared to VSVG control.Accordingly, MV-based transduction is highly specific and efficient invivo.

Example 12 Structural Modifications of the Basic CAR ArchitectureSignificantly Improve Transduction

CD4+ cells were transduced with a “standard” CAR comprising an scFv, CD8hinge, TM CD8, 4-1 BB, and CD3 zeta domains; “modified 1” CAR comprisingan scFv, NGFR stem, NGFR transmembrane, 4-1BB, and CD3 zeta domains; or“modified 2” CAR comprising an scFv, NGFR Cys domain, NGFR stem, NGFRtransmembrane, 4-1 BB, and CD3 zeta domains. Transduction was measuredand showed increase in cells transduced with modified 1 and modified 2CAR constructs, as compare to the standard CAR. Accordingly, structuralmodifications of the basic CAR architecture significantly improvetransduction.

Example 13 Nipah-Based CAR-T Cells Show Similar Activity to VSVG-BasedCAR-T Cells

Cells were transduced with the VSVG vector or the Nipah CAR vector.Expression of CD107, GM-CSF, or IFN-gamma was measured by flowcytometry. Cells transduced with the Nipah CAR vector showed similarexpression patterns of all 3 markers as compared to VSVG transducedcells. Accordingly, Nipah-based CAR-T cells show similar activity toVSVG-based CAR-T cells.

Example 14

HEK293 cells were transfected with plasmid A plus B or plasmid C(control) to produce NiV or VSV-G pseudotyped virus. Expression of GFP,BFP and mScarlet (RFP) were analyzed by flow cytometry 48 hours later.Co-expression of plasmid A plus plasmid B (i.e, a split vector system)reconstituted mScarlet expression via protein splicing mediated by theinteins present in the expressed polypeptides. Cells were also shown toexpress GFP and BFP. Flow cytometry demonstrated that the mScarlet wasproduced, demonstrating that the same cells were transfected with thetwo viral vectors and that the inteins functioned to produce themScarlet.

OTHER EMBODIMENTS

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed:
 1. An engineered pseudotyped lentiviral vectorcomprising: i. an engineered envelope comprising a polypeptide havingthe amino acid sequence of SEQ ID NO: 9; and ii. a heterologouspolypeptide targeting moiety for binding to a target cell.
 2. Theengineered pseudotyped lentiviral vector of claim 1, wherein thetargeting moiety is a scFv, an antigen binding domain, a DARPIN, a VHH,or a FN3 domain.
 3. The engineered pseudotyped lentiviral vector ofclaim 2, wherein the targeting moiety binds to CD7.
 4. The engineeredpseudotyped lentiviral vector of claim 2, wherein the targeting moietybinds to CD8.
 5. The engineered pseudotyped lentiviral vector of claim2, wherein the targeting moiety binds to a protein selected from thegroup consisting of cKit (CDl 17), CD4, CD3, CD3D, CD3E, CD3G, CD3Z,CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34,CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, and CXCR3.
 6. The engineered pseudotyped lentiviral vectorof claim 1, wherein the vector further comprises a nucleic acid moleculeencoding a chimeric antigen receptor (CAR).
 7. The engineeredpseudotyped lentiviral vector of claim 6, wherein the chimeric antigenreceptor comprises an extracellular domain, transmembrane domain, and anintracellular signaling domain.
 8. The engineered pseudotyped lentiviralvector of claim 7, wherein the extracellular domain binds to CD20. 9.The engineered pseudotyped lentiviral vector of claim 7, wherein theextracellular domain binds to CD22, CD123, CD38, CD19, BCMA, CD33, orCD79b.
 10. The engineered pseudotyped lentiviral vector of claim 1,wherein the targeting moiety binds to a target on a Tcell, a CD4+ Tcell,a CD8+ Tcell, a NK cell, an alpha-beta T cell, a gamma-delta Tcell, alymphoid progenitor cell, a hematopoietic stem cell, a myeloid cell, amonocyte, a macrophage, a central memory T cell, a naive T cells, anactivated Tcell, a regulatory T Cell (Treg), or a T-Cell^(CD8+CCR7+).11. The engineered pseudotyped lentiviral vector of claim 1, wherein thetargeting moiety binds to a target on a T cell.
 12. The engineeredpseudotyped lentiviral vector of claim 11, wherein the targeting moietybinds to CD7 on the T cell.
 13. The engineered pseudotyped lentiviralvector of claim 11, wherein the targeting moiety binds to CD8 on the Tcell.
 14. An engineered pseudotyped lentiviral vector comprising anengineered envelope comprising a polypeptide having the amino acidsequence of SEQ ID NO: 9, a targeting moiety that binds to CD7 or CD8 ona T cell and a nucleic acid molecule encoding a chimeric antigenreceptor (CAR), wherein the chimeric antigen receptor comprises anextracellular domain, transmembrane domain, and an intracellularsignaling domain.
 15. The engineered pseudotyped lentiviral vector ofclaim 14, wherein the extracellular domain binds to CD20.
 16. Anengineered pseudotyped lentiviral vector comprising an engineeredenvelope comprising a polypeptide that is at least 90% identical to apolypeptide having the amino acid sequence of SEQ ID NO: 9, a targetingmoiety that binds to CD7 or CD8 on a T cell, and a nucleic acid moleculeencoding a chimeric antigen receptor (CAR), wherein the chimeric antigenreceptor comprises an extracellular domain, transmembrane domain, and anintracellular signaling domain, wherein the extracellular domain bindsto CD20.
 17. The engineered pseudotyped lentiviral vector of claim 16,wherein the engineered envelope comprising a polypeptide that is atleast 95% identical to a polypeptide having the amino acid sequence ofSEQ ID NO:
 9. 18. The engineered pseudotyped lentiviral vector of claim16, wherein the engineered envelope comprising a polypeptide that is atleast 99% identical to a polypeptide having the amino acid sequence ofSEQ ID NO:
 9. 19. A method of infecting a cell in a subject, the methodcomprising administering the engineered pseudotyped lentiviral vector ofclaim 1 to the subject.
 20. A method of infecting a cell in a subject,the method comprising administering the engineered pseudotypedlentiviral vector of claim 16 to the subject.